1 00:00:01 --> 00:00:03 Good morning, class. Nice to be here with you again 2 00:00:03 --> 00:00:07 after a long hiatus. So, we're going to talk today about 3 00:00:07 --> 00:00:11 the cell cycle. That is to say the life of a cell, 4 00:00:11 --> 00:00:15 how a cell grows and divides. And, as it turns out, 5 00:00:15 --> 00:00:18 this is one of our first entrees into cell biology. 6 00:00:18 --> 00:00:22 That is to say we're move, beginning to move away from the 7 00:00:22 --> 00:00:26 molecules inside cells and beginning to look at a, at the next higher 8 00:00:26 --> 00:00:30 level of complexity which is how cells proliferate. 9 00:00:30 --> 00:00:34 It turns out this is a very critical issue in a number of different 10 00:00:34 --> 00:00:38 disease areas, including notably cancer which we'll 11 00:00:38 --> 00:00:42 start talking about next time. And this is also the transition 12 00:00:42 --> 00:00:46 point, the inflection point in the semester when we begin to apply some 13 00:00:46 --> 00:00:50 of the things we've learned earlier to learning about specific disease 14 00:00:50 --> 00:00:54 processes. So, you'll sense more and more 15 00:00:54 --> 00:00:58 introduction of discussions of human disease processes as the semester 16 00:00:58 --> 00:01:02 goes from here on to the end. And here, in fact, 17 00:01:02 --> 00:01:06 is what we call the cell cycle. And I'm going to get into the 18 00:01:06 --> 00:01:10 details of it quite shortly, but the cell cycle is effectively 19 00:01:10 --> 00:01:14 the life cycle of a cell from the moment it is born to the moment that 20 00:01:14 --> 00:01:18 it, in turn, becomes the mother of two daughter cells. 21 00:01:18 --> 00:01:22 And this process is divided up, as you can see, into four different 22 00:01:22 --> 00:01:26 periods here, or as they are called phases. These are cell cycle phases. 23 00:01:26 --> 00:01:30 The first phase is called M or mitosis. 24 00:01:30 --> 00:01:33 The second phase is called G1 phase. The third is called the S or the 25 00:01:33 --> 00:01:37 synthetic phase, and that refers to the fact that 26 00:01:37 --> 00:01:41 this is the time when cells replicate their DNA. 27 00:01:41 --> 00:01:45 And, as you can see, it actually takes a long time 28 00:01:45 --> 00:01:48 because this entire cycle in cultured mammalian cells normally 29 00:01:48 --> 00:01:52 takes as much as a day for a mammalian cell to divide, 30 00:01:52 --> 00:01:56 grow and divide. Contrast that, by the way, with many bacterial 31 00:01:56 --> 00:02:00 cells which can grow and divide in 30, 30 minutes. 32 00:02:00 --> 00:02:04 So, our cells take a lot longer to divide, many of them, 33 00:02:04 --> 00:02:08 but there are exceptions to that. After cells have gone through G, 34 00:02:08 --> 00:02:13 through S phase they go into G2. And these two Gs are, are called Gs 35 00:02:13 --> 00:02:17 because of the gaps they have. Here's the gap between S phase and 36 00:02:17 --> 00:02:22 M phase, or mitosis. G1 is the gap between mitosis and, 37 00:02:22 --> 00:02:26 and the, the subsequent S phase. And, obviously, 38 00:02:26 --> 00:02:31 the most important parts of the cell cycle are the time when the cell 39 00:02:31 --> 00:02:35 divides, which is mitosis, and the time it replicates its DNA 40 00:02:35 --> 00:02:40 which is in S phase. One reason why it's so important is 41 00:02:40 --> 00:02:44 that one has to be sure that cells that arise from the process of 42 00:02:44 --> 00:02:48 growth and division, which is what this is, 43 00:02:48 --> 00:02:52 actually end up with a full genome's worth of DNA and chromosomes. 44 00:02:52 --> 00:02:56 And, therefore, during S phase there has to be a precise 45 00:02:56 --> 00:03:00 replication of all the 3. billion bases in the haploid genome, 46 00:03:00 --> 00:03:05 or 6.4 billion in the diploid genome. 47 00:03:05 --> 00:03:08 And during M phase there has to be a precise allocation of the resulting 48 00:03:08 --> 00:03:12 replicated DNA in equal, exactly equal parts to the two 49 00:03:12 --> 00:03:16 daughter cells. And this is no mean feat. 50 00:03:16 --> 00:03:20 It is an extraordinarily challenging process, 51 00:03:20 --> 00:03:23 which cells, it turns out, with very high efficiency through a 52 00:03:23 --> 00:03:27 variety of complex control mechanisms. And with this outline 53 00:03:27 --> 00:03:31 in mind, I just want to go into some of its details to illustrate to you 54 00:03:31 --> 00:03:35 why we think that the scheme is organized the way it is. 55 00:03:35 --> 00:03:39 The fact of the matter is that the, that the M phase, or the mitosis is 56 00:03:39 --> 00:03:43 actually organized into a series of sub phases. And you may remember 57 00:03:43 --> 00:03:47 that from high school biology but I'll just go into it in some detail, 58 00:03:47 --> 00:03:51 not because we're going to dwell on it but just because I want to give 59 00:03:51 --> 00:03:55 you a feeling for what it's composed of. It's composed of a series of, 60 00:03:55 --> 00:03:59 of sub, sub phases I just mentioned. And, importantly, we start out with 61 00:03:59 --> 00:04:04 an interphase cell. And interphase refers to the 62 00:04:04 --> 00:04:08 entirety of the cell cycle outside of M. That is the entirety, 63 00:04:08 --> 00:04:12 entirety outside of the M phase thereby including G1S and G2. 64 00:04:12 --> 00:04:16 And you notice that what happens in prophase is, first of all, 65 00:04:16 --> 00:04:21 the chromosomes condense. And that's very important because prior 66 00:04:21 --> 00:04:25 to that condensation the chromatin, and chromatin represents the 67 00:04:25 --> 00:04:29 material out of which the chromosomes is assembled consisting 68 00:04:29 --> 00:04:33 largely of DNA and proteins with a big of RNA, the chromatin is 69 00:04:33 --> 00:04:37 dispersed through the nucleus. It's not condensed together. 70 00:04:37 --> 00:04:41 And as such, as you can see here, one doesn't see any specific 71 00:04:41 --> 00:04:44 chromosomes because they're just scattered about the whole 72 00:04:44 --> 00:04:47 chromosomes in a very dilute form. But once the condensations happen, 73 00:04:47 --> 00:04:51 the condensation of chromosomes happen then one begins to appreciate 74 00:04:51 --> 00:04:54 that there are chromosomes. Indeed, if it were not for the case 75 00:04:54 --> 00:04:58 that this, this condensation would happen, it would have taken a very 76 00:04:58 --> 00:05:01 long time before we even realized at the beginning of the 20th century 77 00:05:01 --> 00:05:05 that there were distinct chromosomes themselves. 78 00:05:05 --> 00:05:09 Because when you look in the nucleus like this, and as you, 79 00:05:09 --> 00:05:13 one might do, be doing here stains for DNA, one doesn't see any 80 00:05:13 --> 00:05:17 distinct structures within the nucleus. Here this condensation 81 00:05:17 --> 00:05:22 causes a delineation of these structures in humans, 82 00:05:22 --> 00:05:26 as you know this being 46. And, by the way, this, this, 83 00:05:26 --> 00:05:30 this sequence of events is, is basically standard hardware for all 84 00:05:30 --> 00:05:34 eukaryote cells. It's not as if we invented this 85 00:05:34 --> 00:05:38 recently. This whole sequence of events has largely been around for 1. 86 00:05:38 --> 00:05:42 billion years. One thing you begin to notice here 87 00:05:42 --> 00:05:46 already as we go into the Metaphase, this is the beginning of metaphase, 88 00:05:46 --> 00:05:49 it's sometimes called prometaphase if one wants to split it up even 89 00:05:49 --> 00:05:53 further, is the beginning of the disappearance of the nuclear 90 00:05:53 --> 00:05:57 membrane. So, here we are in a situation where the 91 00:05:57 --> 00:06:01 chromosomes are no longer physically separated from the cytoplasm by the 92 00:06:01 --> 00:06:04 nuclear membrane. The membrane, in effect, 93 00:06:04 --> 00:06:08 disappears. It dissolves. Obviously, at another point it's 94 00:06:08 --> 00:06:12 going to have to reassemble when one starts making daughter cells. 95 00:06:12 --> 00:06:16 And at this point one begins to see quite distinctly the, 96 00:06:16 --> 00:06:19 that, that each chromosome is composed of two chromatids. 97 00:06:19 --> 00:06:23 A chromatid is each one of these arms. And the fact that there are 98 00:06:23 --> 00:06:27 two of them is a direct consequence of the previous DNA replication 99 00:06:27 --> 00:06:31 which made from one chromatid two chromatids. 100 00:06:31 --> 00:06:34 So, these two chromatids are identical, one to the other. 101 00:06:34 --> 00:06:38 They're the consequence of the previous S phase of, 102 00:06:38 --> 00:06:42 the previous DNA replication. And if all things have gone well 103 00:06:42 --> 00:06:46 then these two chromatids have identical DNA sequences because 104 00:06:46 --> 00:06:50 they're the descendants of a single chromatid that existed, 105 00:06:50 --> 00:06:54 actually a chromosome as it's called at that point, 106 00:06:54 --> 00:06:58 existed at the beginning of S phase. In fact, the fidelity of DNA 107 00:06:58 --> 00:07:02 replication, that is to say the accuracy with which DNA is 108 00:07:02 --> 00:07:06 replicated is so good that often one can demonstrate that the mistakes 109 00:07:06 --> 00:07:10 that are made are less than one in ten to the ninth bases. 110 00:07:10 --> 00:07:14 That's stunning. One in ten to the ninth, 111 00:07:14 --> 00:07:18 only one in ten to the ninth basis actually is, ends up being miscopies. 112 00:07:18 --> 00:07:22 In fact, we now realize that the reason why there's such enormous 113 00:07:22 --> 00:07:26 fidelity is that the DNA polymerases that previously begin to replicate 114 00:07:26 --> 00:07:30 the DNA to generate these chromatids make a mistake of about one in ten 115 00:07:30 --> 00:07:35 to the minus five bases. So, they're much more error prone. 116 00:07:35 --> 00:07:39 But there are subsequent proofreading mechanisms. 117 00:07:39 --> 00:07:43 Just the way you would copy edit text, there are proofreading 118 00:07:43 --> 00:07:48 mechanism which erase those errors and reduce the end result of 119 00:07:48 --> 00:07:52 miscopying, of misreplication down in one, down to one and ten to the 120 00:07:52 --> 00:07:56 minus ninth. And given that the genome itself is only, 121 00:07:56 --> 00:08:01 the haploid genome is only 3. billion, that is 3.2 times ten to 122 00:08:01 --> 00:08:05 the ninth basis of DNA, that means that there are only about, 123 00:08:05 --> 00:08:09 roughly speaking, three mistakes made in the entire 124 00:08:09 --> 00:08:14 genome each time a cell goes through a cell cycle in the haploid genome. 125 00:08:14 --> 00:08:18 Now, what we begin to notice here in prometaphase is also the beginning 126 00:08:18 --> 00:08:22 of the assembly of the machinery that will eventually divide those 127 00:08:22 --> 00:08:26 two chromatids from one another. And here, in the midst of metaphase, 128 00:08:26 --> 00:08:30 as you see in the top left, now the chromosomes line up in what 129 00:08:30 --> 00:08:35 is effectively a two-dimensional plane in the middle. 130 00:08:35 --> 00:08:39 It's called an equatorial plane in the middle of the cell. 131 00:08:39 --> 00:08:43 Where, as you can see here in this schematic, each chrome, 132 00:08:43 --> 00:08:47 each chromosome is, here's once again the two chromatids, 133 00:08:47 --> 00:08:51 and these chromid, the places where these two chromatids are held 134 00:08:51 --> 00:08:55 together are lined up in this plane with the arms of the chromosomes 135 00:08:55 --> 00:08:59 kind of flopping around, as you can see here. What's 136 00:08:59 --> 00:09:03 critical here is the points where these, these two chrome, 137 00:09:03 --> 00:09:08 chromatids are joint. And those, those points of joining, 138 00:09:08 --> 00:09:12 as I'll mention in a moment, those are called centromeres. 139 00:09:12 --> 00:09:16 Here, here is a more schematized view of what's about to happen, 140 00:09:16 --> 00:09:20 just to give you a feeling for what this really looks like. 141 00:09:20 --> 00:09:25 And here, as, as we begin to move later into the mitosis, 142 00:09:25 --> 00:09:29 we begin to see that each of the chromatids is joined here by a 143 00:09:29 --> 00:09:33 centromere and that there, and that there are mitotic spindles 144 00:09:33 --> 00:09:37 which are reaching from these paired chromosomes to centrioles at either 145 00:09:37 --> 00:09:42 side of the cell. So, these two centrioles, 146 00:09:42 --> 00:09:46 and the whole apparatus itself is called a centrosome, 147 00:09:46 --> 00:09:51 have microtubules that reach from, from the centro, from the 148 00:09:51 --> 00:09:55 centromeres here in the chromosomes, so the, so the structure in the 149 00:09:55 --> 00:10:00 middle of the chromosome is called a centromere. 150 00:10:00 --> 00:10:05 Via microtubules that reach all the way over to one pole or the other. 151 00:10:05 --> 00:10:10 So, you see, one effect has two poles here. And if, 152 00:10:10 --> 00:10:15 and if we begin to develop this further, we, we've just gotten into 153 00:10:15 --> 00:10:20 serious metaphase here. And now what we see in anaphase is 154 00:10:20 --> 00:10:25 that the two chromatids are being separated apart, 155 00:10:25 --> 00:10:30 they're being pulled apart by these microtubule spindles. 156 00:10:30 --> 00:10:34 These are called spindle fibers, which reach from each of those 157 00:10:34 --> 00:10:38 centrosomes over here back to the chromosomes, and literally are 158 00:10:38 --> 00:10:42 involved in the mechanical pulling them apart, one from the other. 159 00:10:42 --> 00:10:46 And to state the obvious, if we're talking about the fidelity of DNA 160 00:10:46 --> 00:10:50 replication, here we need also to talk about the fact that when one 161 00:10:50 --> 00:10:54 has a set of paired chromatids, one of the chromatids has to go to 162 00:10:54 --> 00:10:58 one side and one to the other. That, in itself, is in principle 163 00:10:58 --> 00:11:02 also an error-prone mechanism, or an error-prone process. 164 00:11:02 --> 00:11:05 Mistakes could be made. And indeed in cancer cells where 165 00:11:05 --> 00:11:09 many of these processes break down sometimes one has both chromatids 166 00:11:09 --> 00:11:13 going to one or the other side. And as you can intuit from that, 167 00:11:13 --> 00:11:16 that leads obviously to a disruption of the normal makeup of the 168 00:11:16 --> 00:11:20 chromosomes that are eventually allocated to each of the two 169 00:11:20 --> 00:11:24 daughter cells. In fact, this allocation happens 170 00:11:24 --> 00:11:28 here at telophase, the last of the four major sub 171 00:11:28 --> 00:11:31 phases of mitosis. Here you see that what's happened is 172 00:11:31 --> 00:11:35 that the, these chromatids have now been pulled apart. 173 00:11:35 --> 00:11:39 And the moment that they're pulled apart and no longer paired up one 174 00:11:39 --> 00:11:42 with the other they now are recognized, once again, 175 00:11:42 --> 00:11:46 to be card-carrying chromosomes. So, now here, here you see the 176 00:11:46 --> 00:11:50 nomenclature has changed. Now they're chromosomes. They're 177 00:11:50 --> 00:11:53 being pulled, pulled apart. And, obviously, one wants an 178 00:11:53 --> 00:11:57 identical set of chromosomes in both daughter cells. 179 00:11:57 --> 00:12:01 And once they're allocated to the two future daughter cells the entire 180 00:12:01 --> 00:12:05 mitotic spindle dissolve. And, as you can imagine, 181 00:12:05 --> 00:12:09 there is a reformation, as is indicated here schematically and not 182 00:12:09 --> 00:12:13 very clearly, in the nuclear membrane. So now the whole cell is 183 00:12:13 --> 00:12:17 reconstructed and now each of the two daughter cells is able, 184 00:12:17 --> 00:12:21 in principle, to split off one from the other and to go off on its merry 185 00:12:21 --> 00:12:25 way. And this looks like a really neat process. And in some 186 00:12:25 --> 00:12:29 eukaryotic cells it happens much more rapidly than the 24 hours that 187 00:12:29 --> 00:12:34 I just indicated to you. There are indications that in some 188 00:12:34 --> 00:12:38 lymphocyte populations it only takes three or four hours. 189 00:12:38 --> 00:12:42 And in some early embryonic cell populations, where there's an 190 00:12:42 --> 00:12:46 absolutely frenetic pace of cell growth and division, 191 00:12:46 --> 00:12:50 it may happen every 30 minutes rather than the 24 hours I just 192 00:12:50 --> 00:12:54 talked about. And you'll say, so what, that, who cares about that. 193 00:12:54 --> 00:12:58 But 30 minutes for replicating an entire 6.4 billion bases of DNA and 194 00:12:58 --> 00:13:02 then allocating them to two daughter cells is actually quite 195 00:13:02 --> 00:13:05 an achievement. And we don't really understand how 196 00:13:05 --> 00:13:09 that happens. I've talked implicitly about the process of cell 197 00:13:09 --> 00:13:13 growth and division. And what I mean to say in more 198 00:13:13 --> 00:13:17 detail about that is the following. After one gets these two initial 199 00:13:17 --> 00:13:21 daughter cells they're obviously only half the size of the previously 200 00:13:21 --> 00:13:25 existing mother cell. And, therefore, what must happen in 201 00:13:25 --> 00:13:29 the subsequent cell cycle is that each of these daughter cells must 202 00:13:29 --> 00:13:33 actually physically grow in size so that it once again becomes a big 203 00:13:33 --> 00:13:37 mama over here after the next cell cycle. 204 00:13:37 --> 00:13:40 And so that growth begins immediately. And much of it occurs 205 00:13:40 --> 00:13:44 just through the generation of new sets of ribosomes, 206 00:13:44 --> 00:13:47 new proteins, new membranes in the cell. All of the complex 207 00:13:47 --> 00:13:51 constituents of a cell need to be duplicated for the next cell cycle 208 00:13:51 --> 00:13:54 including, obviously, all of the organelles, 209 00:13:54 --> 00:13:58 the small structures in the cytoplasm and including, 210 00:13:58 --> 00:14:02 indeed, the mitochondria. So, imagine now the complexity of 211 00:14:02 --> 00:14:06 this because not only is the eukaryotic cell so complex but its 212 00:14:06 --> 00:14:11 entire contents must be faithfully replicated during the subsequent 213 00:14:11 --> 00:14:15 cell cycle. And that obviously implies the intervention, 214 00:14:15 --> 00:14:19 the involvement of very complex control mechanisms about which we 215 00:14:19 --> 00:14:24 understand almost nothing. Now, how, let's get back to this 216 00:14:24 --> 00:14:28 for a moment, because what I've been talking about over the last six to 217 00:14:28 --> 00:14:33 eight minutes is simply mitosis, the M phase here. 218 00:14:33 --> 00:14:37 In fact, in many of these early embryonic cell cycles, 219 00:14:37 --> 00:14:41 what happens is that cells go from M phase directly into S phase and from 220 00:14:41 --> 00:14:45 S phase directly into M. In other words, they don't indulge 221 00:14:45 --> 00:14:49 themselves in a period over here, a long period, and in some cultured 222 00:14:49 --> 00:14:53 mammalian cells this can be 12 or 14 hours of getting set, 223 00:14:53 --> 00:14:58 getting prepared for DNA replication. 224 00:14:58 --> 00:15:01 Similarly, after cells have replicated their DNA, 225 00:15:01 --> 00:15:05 and as we just said, for example, converted one chromosome into two 226 00:15:05 --> 00:15:09 paired chromatids, after they get through S phase 227 00:15:09 --> 00:15:13 they'll also wait another four or five hours, most cultured mammalian 228 00:15:13 --> 00:15:16 cells before they go back into M phase, these two gap periods. 229 00:15:16 --> 00:15:20 Well, how do we actually know how long each of these gaps takes? 230 00:15:20 --> 00:15:24 Well, as is usual, I'm glad I asked that question. 231 00:15:24 --> 00:15:28 And one way we can do so is the following. 232 00:15:28 --> 00:15:32 What we can do is we can take cells and we can block them in mitosis. 233 00:15:32 --> 00:15:37 How can we block them in mitosis? We can use a microtubule antagonist. 234 00:15:37 --> 00:15:42 So, I told you before that the spindle fibers that pull the 235 00:15:42 --> 00:15:47 chromosomes apart are made of microtubules. Microtubules happen 236 00:15:47 --> 00:15:52 to be one of the constituents of the cytoskeleton that gives structure to 237 00:15:52 --> 00:15:57 the cell, and these microtubules are used as the fibers for pulling apart 238 00:15:57 --> 00:16:02 the two chromatids during anaphase, as I just mentioned. 239 00:16:02 --> 00:16:06 And we can use a microtubule antagonist. And in biochemistry, 240 00:16:06 --> 00:16:10 when you want to antagonize something often you use a symbol 241 00:16:10 --> 00:16:15 like this. And, for instance, here's a drug called 242 00:16:15 --> 00:16:19 colcemid, and colcemid is a microtubule antagonist. 243 00:16:19 --> 00:16:23 And even though it has a bit of an effect on the overall cytoskeleton, 244 00:16:23 --> 00:16:28 if you put colcemid on cells then they'll go into mitosis like this 245 00:16:28 --> 00:16:32 and they'll go into metaphase, as we indicated before, but the 246 00:16:32 --> 00:16:37 subsequent sub phases of mitosis will, will grind to a halt. 247 00:16:37 --> 00:16:41 Why? Because the microtubule spindle fibers will be unable to 248 00:16:41 --> 00:16:46 assemble properly because of this, now, I forget whether there's an E 249 00:16:46 --> 00:16:50 here or not. Probably not. OK. Typo. The colcemid prevents 250 00:16:50 --> 00:16:55 the spindle fibers from forming, so therefore cells get hung up, they 251 00:16:55 --> 00:17:00 get blocked in M phase. In fact, if you're interested in 252 00:17:00 --> 00:17:04 looking at the, at the array of chromosomes in a 253 00:17:04 --> 00:17:08 cell, the simplest way of doing it with a mammalian, 254 00:17:08 --> 00:17:12 with a human cell is to put colcemid in it, and the colcemid will block 255 00:17:12 --> 00:17:16 them in, in M phase, they'll block them in, 256 00:17:16 --> 00:17:20 in metaphase, they can't get out of metaphase simply because these pink 257 00:17:20 --> 00:17:24 fibers here are unable to assemble properly, and therefore they get 258 00:17:24 --> 00:17:29 hung up right here while the chromosomes are condensed. 259 00:17:29 --> 00:17:33 And this allows you to visualize the chromosomes under the microscope. 260 00:17:33 --> 00:17:37 Why do need to do that? Well, in the absence of blocking cells and 261 00:17:37 --> 00:17:41 trapping them in, in metaphase, cells will just spin 262 00:17:41 --> 00:17:45 right around the cell cycle and they'll only be in metaphase for 263 00:17:45 --> 00:17:50 maybe only 10 or 15 minutes out of the 24 hours. And, 264 00:17:50 --> 00:17:54 therefore, in a population of cells which is moving around the cell 265 00:17:54 --> 00:17:58 cycle willy-nilly very rapidly, only a minute fraction of the cells 266 00:17:58 --> 00:18:02 at any one point in time will, by chance, be in metaphase when you 267 00:18:02 --> 00:18:07 want to study their chromosomes. So, the best thing to do is to try 268 00:18:07 --> 00:18:12 to block them in metaphase. Now, what you can do, in fact, 269 00:18:12 --> 00:18:16 is put cells in metaphase, and I'm going to draw the cell cycle around 270 00:18:16 --> 00:18:21 here again. Here's M, here's S phase, slightly different 271 00:18:21 --> 00:18:26 than what we just saw before, here's G1, and here is G2. So, 272 00:18:26 --> 00:18:31 now we're going to block cells with colcemid right here in metaphase. 273 00:18:31 --> 00:18:35 And now what happens is that we put metaphase in. Ideally, 274 00:18:35 --> 00:18:39 if we put it in for a day we'd start out with a population of cells which 275 00:18:39 --> 00:18:43 we will call asynchronous. Asynchronous means that at any one 276 00:18:43 --> 00:18:47 point in time these cells will be distributed randomly around the cell 277 00:18:47 --> 00:18:51 cycle. They'll be all over the place, obviously, 278 00:18:51 --> 00:18:55 because there's nothing to cause them to move synchronously through 279 00:18:55 --> 00:18:59 the cell cycle. A random population of cells will be 280 00:18:59 --> 00:19:03 scattered all over the cells cycle like this. We put in colcemid, 281 00:19:03 --> 00:19:07 and what's gradually going to happen is that cells are moving around the 282 00:19:07 --> 00:19:11 cell cycle and when they get to here they'll start piling up. 283 00:19:11 --> 00:19:15 In principle, if all the cells are scattered around the cell cycle at 284 00:19:15 --> 00:19:19 the moment you put in the colcemid, and it takes them 24 hours to get 285 00:19:19 --> 00:19:23 all the way around the cell cycle, then after a day's worth of colcemid 286 00:19:23 --> 00:19:27 treatment all the cells should have piled up right here. 287 00:19:27 --> 00:19:31 In fact, you can begin to look. What, what's the kinetics with which 288 00:19:31 --> 00:19:35 cells pile up into the M phase? And it looks like this. They go 289 00:19:35 --> 00:19:40 around, they go on piling up, and then at the end of 24 hours they 290 00:19:40 --> 00:19:44 plateau. Why do they plateau at 24 hours? Because by 24 hours the 291 00:19:44 --> 00:19:48 stragglers, which started out right here, will have had time to go all 292 00:19:48 --> 00:19:53 the way around the cell cycle and get trapped in M phase. 293 00:19:53 --> 00:19:57 And that gives you a feeling for how long the entire cell cycle time 294 00:19:57 --> 00:20:02 is, now, or this cycle which I called growth and division. 295 00:20:02 --> 00:20:05 And, again, to emphasize, growth is literally the physical 296 00:20:05 --> 00:20:09 growth of the cell. Division is the production of two 297 00:20:09 --> 00:20:12 daughter cells at mitosis. Now, in principle, we can relieve 298 00:20:12 --> 00:20:16 this metaphase block by getting rid of the colcemid. 299 00:20:16 --> 00:20:19 And now, interestingly enough, we have a population of cells that 300 00:20:19 --> 00:20:23 is emerging from metaphase synchronously. 301 00:20:23 --> 00:20:27 What do I mean by that? I mean to indicate that they're all 302 00:20:27 --> 00:20:30 moving in lockstep. They're all moving at the same time 303 00:20:30 --> 00:20:34 out of the cell cycle, so now there's a whole cohort of 304 00:20:34 --> 00:20:37 cells which starts out right here. They've all left metaphase at the 305 00:20:37 --> 00:20:41 same time. This is not an asynchronous culture. 306 00:20:41 --> 00:20:45 And they start moving around, advancing around the cell cycle 307 00:20:45 --> 00:20:48 together. In principle you'd say, well, they should remain synchronous 308 00:20:48 --> 00:20:52 forever. But the fact is all of these phases of the cell cycle, 309 00:20:52 --> 00:20:55 the times are quasi-stochastic, sometimes go, some cells go through 310 00:20:55 --> 00:20:59 G1 in ten hours, some cells go through cells in 311 00:20:59 --> 00:21:03 eleven hours, some through in eight hours. 312 00:21:03 --> 00:21:07 And, as a consequence, as they move further and further 313 00:21:07 --> 00:21:11 around the cell cycle, the population of cells becomes 314 00:21:11 --> 00:21:15 progressively more asynchronous and they won't really enter into the 315 00:21:15 --> 00:21:20 next M phase totally synchronously. But now we can ask the following 316 00:21:20 --> 00:21:24 question, how long is G1? And the way we can do that is to do 317 00:21:24 --> 00:21:28 another kind of experiment where what we do is we release cells, 318 00:21:28 --> 00:21:32 a population of cells from M phase by removing the colcemid, 319 00:21:32 --> 00:21:37 for example. And now we treat cells, 320 00:21:37 --> 00:21:41 we, we treat aliquots of this synchronously advancing population. 321 00:21:41 --> 00:21:46 Each hour we pluck out some of these cells and we expose them for, 322 00:21:46 --> 00:21:51 let's say, the next hour thereafter to some tritiated thymidine. 323 00:21:51 --> 00:21:55 Tritiated thymidine, as you may recall, H3 thymidine as it's 324 00:21:55 --> 00:22:00 sometimes denoted, is obviously a radiolabeled 325 00:22:00 --> 00:22:05 precursor of DNA. And it's going to get incorporated 326 00:22:05 --> 00:22:09 into the DNA and, and to no other macromolecules in 327 00:22:09 --> 00:22:13 the cell. And so the question is if we expose cells for an hour here to 328 00:22:13 --> 00:22:17 tritiated thymidine, how much tritiated thymidine is 329 00:22:17 --> 00:22:21 going to be incorporated into their DNA? How do we know what's 330 00:22:21 --> 00:22:25 incorporated and how much tritiated thymidine remains unincorporated? 331 00:22:25 --> 00:22:30 We extract the DNA from the cells and we precipitate it in an acid. 332 00:22:30 --> 00:22:33 And when that happens the macromolecules of DNA go to the 333 00:22:33 --> 00:22:37 bottom, they precipitate, and the unincorporated tritiated 334 00:22:37 --> 00:22:41 thymidine, which is still soluble, it has not yet been polymerized into 335 00:22:41 --> 00:22:45 DNA molecules, remains in the acid solution. 336 00:22:45 --> 00:22:49 So, we asked how much acid-precipitable thymidine counts 337 00:22:49 --> 00:22:53 are there here? How many in the next, 338 00:22:53 --> 00:22:57 are made in the next hour with another aliquot of cells? 339 00:22:57 --> 00:23:01 How many in the next hour and so forth? And when we do such an 340 00:23:01 --> 00:23:05 experiment we find that we get a, a curve which looks like this. 341 00:23:05 --> 00:23:08 All of a sudden, here we got no sales labeling, 342 00:23:08 --> 00:23:12 in the second hour none, third, fourth, fifth, sixth, in the seventh 343 00:23:12 --> 00:23:16 hour there are some cells which make DNA, in the eighth hour there are 344 00:23:16 --> 00:23:19 some cells, even more cells making DNA, and by the time they're in the 345 00:23:19 --> 00:23:23 ninth or the tenth hour then we find a high rate of DNA synthesis. 346 00:23:23 --> 00:23:27 Now, in principle we could continue this experiment if we wanted to, 347 00:23:27 --> 00:23:31 if we had enough money for tritiated thymidine, which one does because 348 00:23:31 --> 00:23:34 it's very cheap. And what would find is if we started 349 00:23:34 --> 00:23:38 labeling over here with, for another hour pulse, when I say 350 00:23:38 --> 00:23:41 an hour pulse I mean we just put the tritiated thymidine in at the 351 00:23:41 --> 00:23:44 beginning of this one-hour period way over here, 352 00:23:44 --> 00:23:48 and an hour later we take out the cells and extract their DNA and 353 00:23:48 --> 00:23:51 measure how much DNA has been incorporated in that interval. 354 00:23:51 --> 00:23:54 And if we did a tritiated thymidine pulse over here, 355 00:23:54 --> 00:23:58 we'd begin to see that the rate of DNA synthesis was actually lower, 356 00:23:58 --> 00:24:01 and eventually it would go down, back down to this as the cells were 357 00:24:01 --> 00:24:05 moving asynchronously through the culture. 358 00:24:05 --> 00:24:09 What does that mean? Well, this is the time, 359 00:24:09 --> 00:24:13 obviously these cells are in S phase, these cells were in G1, 360 00:24:13 --> 00:24:18 and these cells have now emerged from S phase into G2 and are no 361 00:24:18 --> 00:24:22 longer making DNA. And on that basis we can actually 362 00:24:22 --> 00:24:27 calculate, we can determine how many hours it takes for cells to move 363 00:24:27 --> 00:24:31 through G1. We can do a similar kind of experiment to figure out how 364 00:24:31 --> 00:24:36 long G2 is, the gap 2, G2 phase is. G2, recall, is the time between the 365 00:24:36 --> 00:24:41 ending of DNA replication and the beginning of mitosis. 366 00:24:41 --> 00:24:46 And how do we do that? Well, we can do an inhibitor of DNA 367 00:24:46 --> 00:24:52 synthesis. So, let's say, here is, 368 00:24:52 --> 00:24:57 I'm going to redraw the cell cycle. Here's S phase, G2, I'm drawing it 369 00:24:57 --> 00:25:02 again, here's M, and here's G1. And now what we can do is we'll add 370 00:25:02 --> 00:25:06 a DNA synthesis inhibitor. An inhibitor of DNA synthesis has 371 00:25:06 --> 00:25:10 no effect, as you could imagine, on M phase. This is one of those 372 00:25:10 --> 00:25:14 things, it's called hydroxyurea. It actually blocks the biosynthesis 373 00:25:14 --> 00:25:19 of the precursors, the, the oxyribonucleoside 374 00:25:19 --> 00:25:23 triphosphate precursors of DNA. And, therefore, you add hydroxyurea 375 00:25:23 --> 00:25:27 to cells and they, their, their DNA replication grinds 376 00:25:27 --> 00:25:31 to a halt. OK. So, let's do that. 377 00:25:31 --> 00:25:35 And we'll, we'll, we'll add hydroxyurea to the cells for 24 378 00:25:35 --> 00:25:39 hours. What's going to happen? What will the distribution of cells 379 00:25:39 --> 00:25:42 be afterwards? Well, in fact, 380 00:25:42 --> 00:25:46 some of the cells, when we first added the hydroxyurea 381 00:25:46 --> 00:25:49 the cells were scattered all around, they were asynchronous. We'll add 382 00:25:49 --> 00:25:53 the hydroxyurea and what we'll find is the following. 383 00:25:53 --> 00:25:57 The cells that were in the middle of S phase when we added the 384 00:25:57 --> 00:26:00 hydroxyurea will be stuck dead in the water. They won't be able to 385 00:26:00 --> 00:26:04 move anymore. So, these cells that were in S phase, 386 00:26:04 --> 00:26:08 the moment we add the hydroxyurea will be trapped right here. 387 00:26:08 --> 00:26:12 They cannot make anymore DNA. And, therefore, they'll be frozen 388 00:26:12 --> 00:26:16 at many points, times, points in time in S phase. 389 00:26:16 --> 00:26:20 The cells that are outside of S phase, they can continue to advance 390 00:26:20 --> 00:26:24 all the way around the cell cycle. And, accordingly, there'll be lots 391 00:26:24 --> 00:26:28 of cells over the next 24 hours that are going to just pile up at the 392 00:26:28 --> 00:26:32 G1-S transition. Why? Well, the hydroxyurea has no effect 393 00:26:32 --> 00:26:36 at all on these other points of the cell cycle. And when these cells 394 00:26:36 --> 00:26:41 try to go, go from G1 into S, they cannot get into S phase because 395 00:26:41 --> 00:26:46 they can't make any DNA so they're trapped right over there. 396 00:26:46 --> 00:26:50 And now we'll take away the hydroxyurea, which is, 397 00:26:50 --> 00:26:55 obviously is, to use the notation I used before, an inhibitor of DNA 398 00:26:55 --> 00:27:00 synthesis. And what we can do now is the following. 399 00:27:00 --> 00:27:04 We're going to ask, after we take out the hydroxyurea 400 00:27:04 --> 00:27:08 we're going to put in colcemid. Why do we want to do that? Well, 401 00:27:08 --> 00:27:12 what we really want to do is to ask, after the cells have reached, have 402 00:27:12 --> 00:27:16 gone through S phase, how soon does a labeled cell move 403 00:27:16 --> 00:27:20 from S phase all the way into M phase? How long does it take? 404 00:27:20 --> 00:27:24 How long is G2? And so what we'll do is the following. 405 00:27:24 --> 00:27:28 We take away the hydroxyurea. This allows all these cells to 406 00:27:28 --> 00:27:32 begin to move out. Is this a fully synchronous culture? 407 00:27:32 --> 00:27:36 Well, actually, no, because some of these cells are over here. 408 00:27:36 --> 00:27:39 There's a whole bunch over here. These are synchronous, but the rest 409 00:27:39 --> 00:27:42 of the ones they're already scattered out, 410 00:27:42 --> 00:27:46 so there's going to be some pioneers over here moving ahead of the 411 00:27:46 --> 00:27:49 phalanx, and they'll be some straggles and then there's going to 412 00:27:49 --> 00:27:53 be a big slug of these cells that are moving as the rear guard ahead. 413 00:27:53 --> 00:27:56 Now, what we're going to do, after we take away the hydroxyurea we're 414 00:27:56 --> 00:28:00 going to add our old friend colcemid. 415 00:28:00 --> 00:28:03 And colcemid, I tried to spell it right this time, 416 00:28:03 --> 00:28:06 colcemid is going to block cells, as we obviously said before, right 417 00:28:06 --> 00:28:09 over here. And then what we're going to do is the following. 418 00:28:09 --> 00:28:12 We're going to look, every hour we're going to take some cells and 419 00:28:12 --> 00:28:16 put them in colcemid, or we'll leave them in colcemid the 420 00:28:16 --> 00:28:19 whole time and every hour we'll take cells out of the Petri dish, 421 00:28:19 --> 00:28:22 which have been in colcemid since they were released from here, 422 00:28:22 --> 00:28:25 and we're going to look at the metaphase cells. 423 00:28:25 --> 00:28:29 And how do we look at the metaphase cells? 424 00:28:29 --> 00:28:33 Well, here's a metaphase cell. Here will be its chromosomes. 425 00:28:33 --> 00:28:37 Obviously, they're chromatids at this point. They're under the 426 00:28:37 --> 00:28:41 microscope. And they start accumulating up here. 427 00:28:41 --> 00:28:45 And each and every hour we're going to take some of these metaphase 428 00:28:45 --> 00:28:49 cells, we'll take them out of the Petri dish, and after we've put them 429 00:28:49 --> 00:28:53 in, or we'll leave them in the Petri dish if you want, 430 00:28:53 --> 00:28:57 and after they're there and they've, they've come over here we'll fix 431 00:28:57 --> 00:29:02 them onto the plate. We'll add some alcohol or something 432 00:29:02 --> 00:29:06 which causes them to stick to the plate, they can't swim away, 433 00:29:06 --> 00:29:11 and then after that we'll add some radioactive emulsion. 434 00:29:11 --> 00:29:15 So, here's, let's imagine here is a cell at the bottom of the plate in 435 00:29:15 --> 00:29:20 metaphase, we're going to add some photographic emulsion on top of that 436 00:29:20 --> 00:29:24 like that. And then what we're going to see is how soon we can 437 00:29:24 --> 00:29:29 detect radioactive metaphase chromosomes. How can we do that? 438 00:29:29 --> 00:29:33 Because each time an electron leaves the tritiated thymidine, 439 00:29:33 --> 00:29:38 each time a beta particle leaves it's going to cause a grain of 440 00:29:38 --> 00:29:42 emulsion, of silver to form in the, in the photographic emulsion that 441 00:29:42 --> 00:29:47 we've layered above the cell. So, this photo emulsion is only not 442 00:29:47 --> 00:29:53 a way of detecting light but a way of detecting when there, 443 00:29:53 --> 00:29:58 whenever there's radioactivity in, that's being emitted. And what 444 00:29:58 --> 00:30:04 we're going to look for are grains, silver grains that are located above 445 00:30:04 --> 00:30:08 the chromosomes in the microscope. We could look down through this 446 00:30:08 --> 00:30:12 plate, through the emulsion, on the chromosomes we can see them 447 00:30:12 --> 00:30:16 clearly. I've shown you that before, and we're going to ask ourselves the 448 00:30:16 --> 00:30:20 question, when can we begin to associate silver grains with the 449 00:30:20 --> 00:30:24 chromosomes? Because those silver grains must have been incorporated 450 00:30:24 --> 00:30:28 into the chromosomes during the previous S phase, 451 00:30:28 --> 00:30:32 that is when cells were over here. If a cell was over here and we added 452 00:30:32 --> 00:30:36 colcemid and there won't be any, it won't be radioactively labeled. 453 00:30:36 --> 00:30:40 So, what we're really going to ask now is the following. 454 00:30:40 --> 00:30:44 These cells are all advancing through like this, 455 00:30:44 --> 00:30:48 they're advancing into M phase, when does the first radiolabeled 456 00:30:48 --> 00:30:53 cell get, after we release them with hydroxyurea, when does it get, 457 00:30:53 --> 00:30:57 when do we first see cells like this? And the fact is after five or six 458 00:30:57 --> 00:31:01 hours after we've released them from S phase then we begin to see cells 459 00:31:01 --> 00:31:05 like this which have chromosomes on which there is radio, 460 00:31:05 --> 00:31:10 with which there's radioactivity associated. 461 00:31:10 --> 00:31:15 Well, keep in mind that when cells move through the S phase here, 462 00:31:15 --> 00:31:20 this is when they incorporated tritiated thymidine to their DNA. 463 00:31:20 --> 00:31:25 Cells can't incorporate tritiated thymidine here. 464 00:31:25 --> 00:31:30 They can't incorporate it here. They can't incorporate it here. 465 00:31:30 --> 00:31:33 OK? Right. So, we can allow cells to incorporate a 466 00:31:33 --> 00:31:37 little bit of tritiated thymidine, we can freeze them in here, we'll 467 00:31:37 --> 00:31:40 allow, give them a little bit, bit of tritiated thymidine, let it 468 00:31:40 --> 00:31:44 get into the DNA here, and then we'll add hydroxyurea very 469 00:31:44 --> 00:31:47 shortly thereafter and freeze the cells right like this. 470 00:31:47 --> 00:31:51 So, they'll be, they'll be radioactive by virtue of having 471 00:31:51 --> 00:31:55 dwelled here at different times in the S phase. And when we add 472 00:31:55 --> 00:31:58 hydroxyurea then, the cells that were already in S 473 00:31:58 --> 00:32:02 phase and incorporated a bit of tritiated thymidine will 474 00:32:02 --> 00:32:05 remain in S phase. All of the other cells which were, 475 00:32:05 --> 00:32:08 when we added the hydroxyurea over here, they will go all the way 476 00:32:08 --> 00:32:11 around here. They will move all the way around the cell cycle. 477 00:32:11 --> 00:32:14 They might have happened to incorporated a little bit of 478 00:32:14 --> 00:32:17 tritiated thymidine before we added the hydroxyurea, 479 00:32:17 --> 00:32:21 but they'll get trapped over here. So the only, so the cells that have 480 00:32:21 --> 00:32:24 tritiated thymidine, they will be scattered at various 481 00:32:24 --> 00:32:27 points in the S phase. We'll remove the hydroxyurea that 482 00:32:27 --> 00:32:30 allows the cells to escape from S phase and move all the 483 00:32:30 --> 00:32:34 way around to here. And then we begin to look for seeing 484 00:32:34 --> 00:32:40 when cells get radiolabeled chromosomes. Is that clearer now? 485 00:32:40 --> 00:32:45 A little clear. OK. Now, let's begin to ask how this is all 486 00:32:45 --> 00:32:51 coordinated. What determines how cells, when cells will grow and when 487 00:32:51 --> 00:32:57 cells with not grow? So, let's go back to our depiction 488 00:32:57 --> 00:33:03 of the cell cycle. How do, what controls all this? 489 00:33:03 --> 00:33:09 What determines when a cell is going to move around the cell cycle? 490 00:33:09 --> 00:33:15 In our bodies we have roughly three times ten to the thirteenth cells. 491 00:33:15 --> 00:33:21 I don't know if that's a number I mentioned to you before. 492 00:33:21 --> 00:33:27 It's a pretty interesting number. It's probably not the most 493 00:33:27 --> 00:33:32 interesting number. By now far more people are 494 00:33:32 --> 00:33:36 interested in the number of how many games the Red Sox have won or lost, 495 00:33:36 --> 00:33:41 but it's a number anyhow. How many cell divisions do we go through in a 496 00:33:41 --> 00:33:46 lifetime? Did we ever talk about that? How many times do we have a 497 00:33:46 --> 00:33:50 cell growth and division cycle in a human lifetime, 498 00:33:50 --> 00:33:55 lifetime? And the answer is in human lifetime there are about ten 499 00:33:55 --> 00:34:00 to the sixth cell divisions. It's a staggering number. 500 00:34:00 --> 00:34:03 Ten to the sixteenth in one human body in a human lifetime. 501 00:34:03 --> 00:34:07 And if you figure out what that amounts to, I think it's something 502 00:34:07 --> 00:34:11 like, and you can figure it out yourself, I think it amounts to 503 00:34:11 --> 00:34:15 something like ten to the seventh cell divisions in each second. 504 00:34:15 --> 00:34:18 So, each time I'm talking with you I'm going, I've already gone through 505 00:34:18 --> 00:34:22 any one of my sentences. And by the time my longwinded 506 00:34:22 --> 00:34:26 sentence is finished, I've probably already had 100 507 00:34:26 --> 00:34:30 million mitoses happening inside of me. That's -- 508 00:34:30 --> 00:34:33 Maybe even more because some of my sentences are really long. 509 00:34:33 --> 00:34:37 So, imagine that, ten to the seventh for a second. 510 00:34:37 --> 00:34:40 You can figure out the number. This is a lifetime of let's say you 511 00:34:40 --> 00:34:44 live 70 years, you can do the math of how many it 512 00:34:44 --> 00:34:48 is per second. A lot of that's going on, 513 00:34:48 --> 00:34:51 by the way, in the bone marrow and in the gut, because in the bone 514 00:34:51 --> 00:34:55 marrow you're constantly replacing a lot of your red blood cells. 515 00:34:55 --> 00:34:59 You have lots of them, but they only last for 120 days. 516 00:34:59 --> 00:35:02 So, after 120 days a red blood cell is broken down and you get a new red 517 00:35:02 --> 00:35:06 blood cell in its place. But obviously you have more than 518 00:35:06 --> 00:35:10 one blood cell in your body. The same thing happens in the gut. 519 00:35:10 --> 00:35:14 The cells lining the gut, those are called epithelial cells, 520 00:35:14 --> 00:35:18 they're lining the surface of the gut, and they are constantly being 521 00:35:18 --> 00:35:22 sloughed off the wall of the gut. Why? Because it's not so nice to 522 00:35:22 --> 00:35:26 live on the wall of the gut. It's not a very pleasant 523 00:35:26 --> 00:35:29 environment. And the cells are therefore 524 00:35:29 --> 00:35:33 constantly gotten, gotten rid of because they have a 525 00:35:33 --> 00:35:37 hard time surviving for extended periods of time. 526 00:35:37 --> 00:35:41 Those cells live only three or four days on the wall of the gut. 527 00:35:41 --> 00:35:44 So, when you go to the bathroom, I don't mean to be too graphic, but 528 00:35:44 --> 00:35:48 number two, a significant proportion of what comes out the bottom is 529 00:35:48 --> 00:35:52 actually cells that have been sloughed off from the epithelial 530 00:35:52 --> 00:35:56 lining of the gut. I don't know what the proportion is. 531 00:35:56 --> 00:36:00 It's something like 20% or 30%. About half of what comes out is 532 00:36:00 --> 00:36:04 actually bacterial in the, in the feces. We're not having 533 00:36:04 --> 00:36:08 lunch now so you can, you can absorb all this, 534 00:36:08 --> 00:36:12 right? About half of it. Almost none of the bulk of the 535 00:36:12 --> 00:36:16 feces actually ends up being what you ate because most of what you ate 536 00:36:16 --> 00:36:20 ended up, ends up getting compressed to a very small amount of solid 537 00:36:20 --> 00:36:24 matter. So, most of what comes out is, is not what, 538 00:36:24 --> 00:36:28 what came in, is not the processing of food but other things, 539 00:36:28 --> 00:36:32 as I've just mentioned. And, by the way, 540 00:36:32 --> 00:36:37 here's another unsettling statistic. It's not so graphic but it's 541 00:36:37 --> 00:36:42 unsettling if you think about it. There are more bacterial cells in 542 00:36:42 --> 00:36:46 your gut than in the rest, than there are mammalian eukaryotic 543 00:36:46 --> 00:36:51 cells in the rest of your body. They're taking over. There are 544 00:36:51 --> 00:36:56 more of them than there are us in each one of our bodies. 545 00:36:56 --> 00:37:01 Go figure. Anyhow. Why am I going on this long 546 00:37:01 --> 00:37:05 excursion? Well, only he knows and he's not telling. 547 00:37:05 --> 00:37:09 But there was a reason behind it. The reason why I'm going into this 548 00:37:09 --> 00:37:13 is to impress on you the fact that it's really important that the 549 00:37:13 --> 00:37:17 advance of cells through their growth and division cycle that we've 550 00:37:17 --> 00:37:21 just talked about here is very carefully controlled, 551 00:37:21 --> 00:37:25 because each one of these growth, cell growth and division cycles is 552 00:37:25 --> 00:37:30 the opportunity for disaster. What kind of disaster? 553 00:37:30 --> 00:37:33 Well, if the cell makes a mistake in its chromosomes it might die. 554 00:37:33 --> 00:37:37 The death of a cell in our body is not a disaster. 555 00:37:37 --> 00:37:40 I just indicated to you that we're, we're making ten to the seventh new 556 00:37:40 --> 00:37:44 cells every second, but hopefully we're making exactly 557 00:37:44 --> 00:37:48 the same number of cells that die. So, ten to the seventh cells are 558 00:37:48 --> 00:37:51 made for every ten to the seventh cells that die. 559 00:37:51 --> 00:37:55 If you have an excess of new cells compared with dying cells you're in 560 00:37:55 --> 00:37:59 bad shape because that's really the disease of cancer. 561 00:37:59 --> 00:38:02 And that implies, even with our knowing any, 562 00:38:02 --> 00:38:05 without our knowing anything else about cancer, that there have to be 563 00:38:05 --> 00:38:09 very careful controls over the decision on the part of a cell to 564 00:38:09 --> 00:38:12 whether, as to whether it can grow or divide, as to whether or not it 565 00:38:12 --> 00:38:16 should grow and divide. Now, what does that mean? 566 00:38:16 --> 00:38:19 Well, part of the decisions come from the following situation. 567 00:38:19 --> 00:38:22 Let's imagine here we're growing, we're talking about a human tissue. 568 00:38:22 --> 00:38:26 Each one of these things is a cell. And, as usual, my art is not that 569 00:38:26 --> 00:38:30 splendid. But one of the things I want to 570 00:38:30 --> 00:38:34 impress on you is that the body cannot give license to this cell or 571 00:38:34 --> 00:38:38 to this cell or to this cell to make the decision about growing and 572 00:38:38 --> 00:38:42 dividing on its own. A cell cannot go and say I think I 573 00:38:42 --> 00:38:46 feel like dividing, I'm going to go do it. 574 00:38:46 --> 00:38:50 That's a no-no. Why is it a no-no? Because the moment one grants 575 00:38:50 --> 00:38:54 autonomy to such a cell one's in a very dangerous situation. 576 00:38:54 --> 00:38:58 Instead, what has to happen, what must happen is that cells talk 577 00:38:58 --> 00:39:03 to one another and cells read a consensus. 578 00:39:03 --> 00:39:06 For instance, maybe there's a cell that's missing right over here in 579 00:39:06 --> 00:39:10 this tissue. And keep in mind an architecturally complex tissue has a 580 00:39:10 --> 00:39:13 precise number of cells in it organized in a very precise pattern. 581 00:39:13 --> 00:39:17 And the way that pattern is maintained is that the cells are 582 00:39:17 --> 00:39:21 constantly looking around and seeing is there a gap here? 583 00:39:21 --> 00:39:24 Do we need to make a new cell or not? It's not as if this cell says, 584 00:39:24 --> 00:39:28 well, I'm happy here and all my neighbors are happy but, 585 00:39:28 --> 00:39:32 still, I'm going to grow and divide because it seems like 586 00:39:32 --> 00:39:36 a nice thing to do. Again, that's an invitation for 587 00:39:36 --> 00:39:40 disaster. And I'm saying that if only to impress upon you the fact 588 00:39:40 --> 00:39:44 that cells only will make the decision to grow and divide and to 589 00:39:44 --> 00:39:48 go into this cell cycle on the basis of consultations with their 590 00:39:48 --> 00:39:52 neighbors. They're constantly talking to one another. 591 00:39:52 --> 00:39:56 The signals, the intercellular signals within a living tissue 592 00:39:56 --> 00:40:00 represent constant ceaseless chatter. 593 00:40:00 --> 00:40:03 It's like everybody is on a cell phone talking with six or eight or 594 00:40:03 --> 00:40:07 ten of his or her neighbors all the time. And if you could hear it, 595 00:40:07 --> 00:40:11 it would be a real din. So, that raised the question how do cells 596 00:40:11 --> 00:40:15 talk with one another? What messages do they exchange, 597 00:40:15 --> 00:40:18 one with the other, so that they can establish some kind of consensus as 598 00:40:18 --> 00:40:22 to whether or not it's inappropriate for one of their number to grow and 599 00:40:22 --> 00:40:26 divide? All right. This is an unauthorized ad. 600 00:40:26 --> 00:40:30 You can interpret it the way you want. 601 00:40:30 --> 00:40:34 Anyhow. So how do they do that? Well, what they do, one of the most 602 00:40:34 --> 00:40:38 important ways they communicate is they exchange proteins with one 603 00:40:38 --> 00:40:42 another called growth factors. And a growth factor goes from one 604 00:40:42 --> 00:40:46 cell, here's a cell, it releases a growth factor and it 605 00:40:46 --> 00:40:51 goes to a second cell and induces the second cell to begin to divide. 606 00:40:51 --> 00:40:55 That's a mechanism of transferring the signal or conveying the signal. 607 00:40:55 --> 00:40:59 And a growth factor itself is a relatively low molecular weight 608 00:40:59 --> 00:41:03 protein which travels through interstellular, 609 00:41:03 --> 00:41:08 interstellar, intercellular space. It moves from one cell to the other. 610 00:41:08 --> 00:41:12 So, it's secreted by one cell, it moves here, and then it goes over 611 00:41:12 --> 00:41:16 here to a second cell. In fact, if we want to blow up the 612 00:41:16 --> 00:41:20 second cell, the second cell has on its surface a specific protein which 613 00:41:20 --> 00:41:25 is known as a receptor or, in this case, we can say a growth 614 00:41:25 --> 00:41:29 factor receptor, which represents a means by which 615 00:41:29 --> 00:41:33 that cell senses the presence of a growth factor in the extracellular 616 00:41:33 --> 00:41:38 space. And, as is suggested in this very 617 00:41:38 --> 00:41:43 crude and poorly drawn cartoon, this receptor is a transmembrane 618 00:41:43 --> 00:41:48 protein. It has an extra cellular domain, it has an intracellular 619 00:41:48 --> 00:41:54 domain, and it moves through, it protrudes through the lipid 620 00:41:54 --> 00:41:59 bilayer of the plasma membrane. We talked very briefly about these 621 00:41:59 --> 00:42:04 at the beginning of the semester. And what happens is these receptors 622 00:42:04 --> 00:42:08 are so configured that in the event that a growth factor comes over and 623 00:42:08 --> 00:42:13 is sent off by a neighboring cell, the growth factor, I'll draw it here 624 00:42:13 --> 00:42:18 as a, as a square, this growth factor binds to the 625 00:42:18 --> 00:42:22 receptor in a very specific fashion. And, as a consequence, the receptor 626 00:42:22 --> 00:42:27 responds in its intracellular or cytoplasmic domain by emitting 627 00:42:27 --> 00:42:31 signals into the cell informing the cell that an encounter has been had 628 00:42:31 --> 00:42:36 in the extracellular space with this growth factor which has been 629 00:42:36 --> 00:42:41 released ostensibly by another cell. 630 00:42:41 --> 00:42:45 Now, the fact of the matter is there are many different kinds of growth 631 00:42:45 --> 00:42:49 factors. Here I've drawn, drawn, drawn a square one. Here's a 632 00:42:49 --> 00:42:53 triangular one. Here's a circular one. 633 00:42:53 --> 00:42:57 And each one of these growth factors has its own cognate receptor. 634 00:42:57 --> 00:43:02 So, here's a receptor that binds circular growth factors. 635 00:43:02 --> 00:43:06 Here's a receptor that binds triangular growth factors. 636 00:43:06 --> 00:43:10 Well, obviously I'm very schematic. The point I wish to make is that 637 00:43:10 --> 00:43:14 there are multiple distinct kinds of receptors, and each growth factor we 638 00:43:14 --> 00:43:19 can, I'll abbreviate it as a, as a GF, each growth factor is 639 00:43:19 --> 00:43:23 what's called a ligand, or a ligand depending on where you 640 00:43:23 --> 00:43:27 live, a ligand for the receptor. So, in this case the square thing 641 00:43:27 --> 00:43:32 is the ligand for the receptor. It binds to the receptor and, 642 00:43:32 --> 00:43:36 in so doing, it provokes the receptor to emit a signal which his 643 00:43:36 --> 00:43:40 transferred in through the plasma membrane into the cell cytoplasm. 644 00:43:40 --> 00:43:44 Clearly, what I also wish to indicate by this is that growth 645 00:43:44 --> 00:43:49 factors don't bind to inappropriate receptors. They only bind to their 646 00:43:49 --> 00:43:53 appropriate receptors or their cognate receptors. 647 00:43:53 --> 00:43:57 So, for example, there's a growth factor that we'll talk 648 00:43:57 --> 00:44:00 about shortly. It's called epidermal 649 00:44:00 --> 00:44:04 growth factor. 650 00:44:04 --> 00:44:07 And I'll just abbreviate GF for growth factor. 651 00:44:07 --> 00:44:10 There's another growth factor that called platelet-derived 652 00:44:10 --> 00:44:16 growth factor. 653 00:44:16 --> 00:44:20 So, we have EGF, we have PDGF, platelet-derived 654 00:44:20 --> 00:44:24 growth factor. And, in fact, there's actually 30 655 00:44:24 --> 00:44:28 or 40 other kinds of growth factors. And each one binds specifically to 656 00:44:28 --> 00:44:32 its own receptors. And so now we begin to understand 657 00:44:32 --> 00:44:36 that the signaling channels are actually very multiplexed. 658 00:44:36 --> 00:44:40 It's not just at one, one kind of growth factor can travel through 659 00:44:40 --> 00:44:44 intercellular space. And, again, this cell will only 660 00:44:44 --> 00:44:48 receive, will only make a decision to grow and divide if its growth 661 00:44:48 --> 00:44:52 factor receptors have send an adequate number of stimulatory 662 00:44:52 --> 00:44:56 signals into the cell which then pursued the signaling circuitry 663 00:44:56 --> 00:45:00 which processes these signals that it is indeed time to make the binary 664 00:45:00 --> 00:45:04 decision, growth versus non-growth on the basis of these signals 665 00:45:04 --> 00:45:09 received from the extracellular space. 666 00:45:09 --> 00:45:13 From that we can already intuit something interesting about cancer 667 00:45:13 --> 00:45:18 cells. Cancer cells make a decision to grow even without having received 668 00:45:18 --> 00:45:22 the signal from their neighbors. In other words, cancer cells become 669 00:45:22 --> 00:45:27 quasi or totally, they become growth factor 670 00:45:27 --> 00:45:32 independent. That is to say somehow they have 671 00:45:32 --> 00:45:37 usurped and perturbed this signaling mechanism and they have no longer 672 00:45:37 --> 00:45:41 become dependent on these extracellular signals in order to 673 00:45:41 --> 00:45:46 make the decision to grow and divide. They will just grow in essentially 674 00:45:46 --> 00:45:51 an autonomous or independent fashion. And we know a little bit about this 675 00:45:51 --> 00:45:56 from the following kind of experiment. 676 00:45:56 --> 00:46:00 If you put a human connective tissue cell, a connective tissue cell is 677 00:46:00 --> 00:46:05 called a fibroblast in culture or even a mouse, fibroblast in culture, 678 00:46:05 --> 00:46:09 you put it in a Petri dish like this, it'll sit on the bottom. 679 00:46:09 --> 00:46:14 You can provide this fibroblast or fibroblasts with all the nutrients 680 00:46:14 --> 00:46:18 it needs, with glucose and with vitamins and vital amino, 681 00:46:18 --> 00:46:23 essential amino acids, whatever you want, give it all those low 682 00:46:23 --> 00:46:27 molecular weight compounds and the, and the fibroblasts will sit down on 683 00:46:27 --> 00:46:32 the dish. And it will look up at you and smile. 684 00:46:32 --> 00:46:37 And it will do so for days and weeks. It won't do anything. 685 00:46:37 --> 00:46:42 It won't grow at all. Why? Because you haven't given it the 686 00:46:42 --> 00:46:46 requisite signals to induce it, to persuade it, to proliferate. And 687 00:46:46 --> 00:46:51 what are those requisite signals? They are growth factors. So, you 688 00:46:51 --> 00:46:56 need to add growth factors in addition to the nutrients in order 689 00:46:56 --> 00:47:01 to persuade a cell to emerge from its quiescent state into the active 690 00:47:01 --> 00:47:07 growth and division cycle. And so in my last minutes or my last 691 00:47:07 --> 00:47:13 minute, hopefully not on this earth but in this lecture, 692 00:47:13 --> 00:47:19 let me just say that when you, I mean nothing is sure, but if you 693 00:47:19 --> 00:47:25 take a cell and you deprive it, you starve it of growth factors it 694 00:47:25 --> 00:47:31 exits from the active growth and division cycle and it will go out 695 00:47:31 --> 00:47:36 into another phase called G zero. And it will sit there like a bump on 696 00:47:36 --> 00:47:40 a log for an extended period of time. It will be metabolically active, 697 00:47:40 --> 00:47:45 viable, but it won't proliferate. It will be like the cells you put in 698 00:47:45 --> 00:47:50 the Petri dish without growth factors. And if you give it growth 699 00:47:50 --> 00:47:54 factors then the cell will go back into the G1 phase of the cell cycle 700 00:47:54 --> 00:47:59 and begin active growth cycling. It will pass through a successive 701 00:47:59 --> 00:48:04 cycles of growth and division as long as you give it growth factors. 702 00:48:04 --> 00:48:08 And each time the cell emerges from M it's going to ask the question, 703 00:48:08 --> 00:48:13 are there any growth factors around me? If so, I might commit myself to 704 00:48:13 --> 00:48:17 doing this over again. And if there aren't, I'm getting 705 00:48:17 --> 00:48:22 out of this racetrack and going over to G zero where I may stay for days, 706 00:48:22 --> 00:48:26 weeks or years. On that dramatic and tense note, see 707 00:48:26 --> 48:31 you on Wednesday.