1 00:00:16,434 --> 00:00:21,280 ADAM MARTIN: And so today and for the remainder of the week, 2 00:00:21,280 --> 00:00:25,000 the theme is going to be the cell division cycle. 3 00:00:25,000 --> 00:00:27,880 And so we're going to really talk about the cell division 4 00:00:27,880 --> 00:00:32,770 cycle in every lecture this week with the penultimate lecture 5 00:00:32,770 --> 00:00:37,720 talking about how dysregulation of the cell division cycle 6 00:00:37,720 --> 00:00:42,170 results in a pathological condition known as cancer. 7 00:00:42,170 --> 00:00:46,130 OK, so here is now a cell going through the cell division 8 00:00:46,130 --> 00:00:46,630 cycle. 9 00:00:46,630 --> 00:00:49,870 It's entered into mitosis right now. 10 00:00:49,870 --> 00:00:54,130 And these guys here are the chromosomes of the cell. 11 00:00:54,130 --> 00:00:57,520 And you're going to see them line up at the metaphase plate. 12 00:00:57,520 --> 00:00:59,920 And eventually they'll be segregated 13 00:00:59,920 --> 00:01:02,270 to the two poles of the cell. 14 00:01:02,270 --> 00:01:05,330 And then the cell will divide along its equator. 15 00:01:05,330 --> 00:01:08,170 OK, so I thought we could start today 16 00:01:08,170 --> 00:01:12,280 by just thinking about what has to happen in a cell 17 00:01:12,280 --> 00:01:16,000 during the cell division cycle. 18 00:01:16,000 --> 00:01:22,450 What has to happen during this process in order for the cell 19 00:01:22,450 --> 00:01:24,040 to replicate? 20 00:01:24,040 --> 00:01:26,430 Yes, Miles? 21 00:01:26,430 --> 00:01:30,896 AUDIENCE: For all the [INAUDIBLE] 22 00:01:30,896 --> 00:01:34,228 all those have to be duplicated so that each cell has 23 00:01:34,228 --> 00:01:35,656 a starting number. 24 00:01:35,656 --> 00:01:38,970 ADAM MARTIN: Mmm hmm. 25 00:01:38,970 --> 00:01:41,230 So Miles suggested the organelles 26 00:01:41,230 --> 00:01:50,140 have to be duplicated such that the daughter cells can inherit 27 00:01:50,140 --> 00:01:51,400 those organelles. 28 00:01:51,400 --> 00:01:53,710 And that's correct. 29 00:01:53,710 --> 00:01:55,690 What else has to happen? 30 00:01:55,690 --> 00:01:57,240 Anything else have to be duplicated? 31 00:01:57,240 --> 00:01:57,740 Stephen-- 32 00:01:57,740 --> 00:01:59,198 AUDIENCE: DNA has to be duplicated. 33 00:01:59,198 --> 00:02:02,050 ADAM MARTIN: The DNA, the nuclear DNA, the chromosomes, 34 00:02:02,050 --> 00:02:03,190 have to be duplicated. 35 00:02:06,810 --> 00:02:08,979 So the chromosomes have to be duplicated-- 36 00:02:14,500 --> 00:02:15,260 duplicated. 37 00:02:20,060 --> 00:02:23,080 What else has to happen every cell cycle? 38 00:02:33,420 --> 00:02:36,960 What would happen to the size of the cell just when it divides? 39 00:02:42,090 --> 00:02:42,742 Yeah, Udo? 40 00:02:42,742 --> 00:02:45,160 AUDIENCE: It would grow. 41 00:02:45,160 --> 00:02:47,310 ADAM MARTIN: So Udo is suggesting that the cell has 42 00:02:47,310 --> 00:02:48,180 to grow, right? 43 00:02:48,180 --> 00:02:51,210 Because if the cell didn't grow, then cell division 44 00:02:51,210 --> 00:02:54,640 would make smaller and smaller and smaller cells. 45 00:02:54,640 --> 00:02:58,980 And so another thing the cell has to do during the cell cycle 46 00:02:58,980 --> 00:03:01,185 at some point, it has to grow in size. 47 00:03:03,950 --> 00:03:07,980 OK, and what's the final point of the cell cycle? 48 00:03:07,980 --> 00:03:08,565 What happens? 49 00:03:11,160 --> 00:03:13,200 What's kind of the goal of the cell cycle? 50 00:03:16,920 --> 00:03:17,670 Yes, Stephen? 51 00:03:17,670 --> 00:03:18,753 AUDIENCE: Undergo mitosis. 52 00:03:18,753 --> 00:03:20,850 ADAM MARTIN: To undergo mitosis. 53 00:03:20,850 --> 00:03:23,670 And so the cell has to physically divide, right? 54 00:03:23,670 --> 00:03:25,890 The chromosomes have to be segregated, 55 00:03:25,890 --> 00:03:29,150 and the cell has to physically divide. 56 00:03:29,150 --> 00:03:34,590 OK, so you can think of the cell cycle 57 00:03:34,590 --> 00:03:38,190 as the goal of getting all these events to happen 58 00:03:38,190 --> 00:03:41,400 is to get one cell to become two cells. 59 00:03:41,400 --> 00:03:45,820 So you need chromosome segregation. 60 00:03:45,820 --> 00:03:50,400 And you want an equal segregation of genetic material 61 00:03:50,400 --> 00:03:53,505 into two daughter cells after the cell divides. 62 00:04:00,330 --> 00:04:03,660 OK, so today, we're going to unpack the mechanisms 63 00:04:03,660 --> 00:04:07,170 that allow a cell to do many of these things 64 00:04:07,170 --> 00:04:08,670 and how it's regulated. 65 00:04:11,760 --> 00:04:14,640 And one thing to think about is, what 66 00:04:14,640 --> 00:04:16,529 is going to determine whether or not 67 00:04:16,529 --> 00:04:21,209 a cell enters the cell division cycle and undergoes a division? 68 00:04:21,209 --> 00:04:23,370 What do you think are some things that cells 69 00:04:23,370 --> 00:04:28,830 would care about if it's trying to decide whether or not 70 00:04:28,830 --> 00:04:29,430 to divide? 71 00:04:33,420 --> 00:04:36,230 So one thing a cell is going to care about in a multicellular 72 00:04:36,230 --> 00:04:39,890 setting is whether it's getting appropriate communications 73 00:04:39,890 --> 00:04:43,010 from other cells that are telling it to divide. 74 00:04:43,010 --> 00:04:44,840 And remember, Professor Imperiali 75 00:04:44,840 --> 00:04:46,370 told you about signaling. 76 00:04:46,370 --> 00:04:50,540 And one example was receptor tyrosine kinase signaling. 77 00:04:50,540 --> 00:04:54,170 And this is just a diagram showing you the RAS map kinase 78 00:04:54,170 --> 00:04:55,400 pathway. 79 00:04:55,400 --> 00:04:59,330 And one of the effects of this signaling pathway 80 00:04:59,330 --> 00:05:03,560 is to promote cells to enter the cell division cycle in order 81 00:05:03,560 --> 00:05:06,320 to divide, OK? 82 00:05:06,320 --> 00:05:09,070 So this is in a multicellular organism, 83 00:05:09,070 --> 00:05:10,880 the signaling is important. 84 00:05:10,880 --> 00:05:14,420 For unicellular organisms, cells might care about whether or not 85 00:05:14,420 --> 00:05:18,320 there are nutrients present or whether or not the cell is 86 00:05:18,320 --> 00:05:21,050 of the right size, OK? 87 00:05:21,050 --> 00:05:23,320 So cells have to make the decision. 88 00:05:23,320 --> 00:05:26,570 I'm going to focus mainly on cell communication 89 00:05:26,570 --> 00:05:31,490 and how that might change the cell physiology. 90 00:05:31,490 --> 00:05:33,260 And I want to start by just giving you 91 00:05:33,260 --> 00:05:36,260 a little bit of an overview of the cell division cycle. 92 00:05:36,260 --> 00:05:38,270 So there are four distinct phases 93 00:05:38,270 --> 00:05:40,670 of the cell division cycle. 94 00:05:40,670 --> 00:05:43,010 And I split them into two classes. 95 00:05:43,010 --> 00:05:47,490 There are phases where things physically happen to the cell. 96 00:05:47,490 --> 00:05:50,420 And those are S phase and M phase. 97 00:05:50,420 --> 00:05:55,790 And so during S phase, S phase stands for DNA synthesis. 98 00:05:59,940 --> 00:06:05,300 And it's during this phase when the nuclear DNA is replicated, 99 00:06:05,300 --> 00:06:06,440 OK? 100 00:06:06,440 --> 00:06:10,310 And so for each of the action phases, if you will, 101 00:06:10,310 --> 00:06:12,020 there's some sort of machinery that's 102 00:06:12,020 --> 00:06:15,260 involved in changing the cell. 103 00:06:15,260 --> 00:06:17,030 In the case of S phase, that would 104 00:06:17,030 --> 00:06:22,610 be DNA polymerase and helicases that mediate the replication. 105 00:06:22,610 --> 00:06:29,150 So helicases, DNA polymerase. 106 00:06:29,150 --> 00:06:32,090 And you remember from earlier in the semester 107 00:06:32,090 --> 00:06:34,310 when we talked about DNA replication 108 00:06:34,310 --> 00:06:36,140 all of the proteins that are involved 109 00:06:36,140 --> 00:06:39,170 in replicating a chromosome. 110 00:06:39,170 --> 00:06:43,370 OK, the other phase where something really physical 111 00:06:43,370 --> 00:06:45,725 happens is M phase, which is mitosis. 112 00:06:48,790 --> 00:06:53,090 And during M phase, this is when the sister chromatids 113 00:06:53,090 --> 00:06:56,930 of each chromosome are separated to the daughter cells. 114 00:06:56,930 --> 00:07:00,260 OK, so this is when chromosome segregation happens. 115 00:07:08,810 --> 00:07:10,950 And again, during this phase, there 116 00:07:10,950 --> 00:07:13,290 has to be some sort of machine that 117 00:07:13,290 --> 00:07:17,160 gets activated at this phase of the cell cycle in order 118 00:07:17,160 --> 00:07:20,310 to, in this case, physically separate the chromosomes 119 00:07:20,310 --> 00:07:21,900 from each other. 120 00:07:21,900 --> 00:07:29,550 And that machine in M phase is the mitotic spindle, 121 00:07:29,550 --> 00:07:35,850 which you'll recall is a machine that consists of microtubules. 122 00:07:35,850 --> 00:07:37,610 So these are microtubules. 123 00:07:43,410 --> 00:07:46,650 And in each cell cycle, these events have to happen. 124 00:07:46,650 --> 00:07:49,080 But they have to happen in order, right? 125 00:07:49,080 --> 00:07:52,930 You need DNA synthesis before you segregate the chromosomes, 126 00:07:52,930 --> 00:07:53,430 right? 127 00:07:53,430 --> 00:07:55,650 So there has to be an order to this. 128 00:08:00,210 --> 00:08:03,375 So these other phases are called gap phases. 129 00:08:06,870 --> 00:08:08,835 And there are two of them, G1 and G2. 130 00:08:11,730 --> 00:08:14,130 And events happen during these gap phases 131 00:08:14,130 --> 00:08:19,560 to help to ensure that things happen in the right order. 132 00:08:19,560 --> 00:08:23,910 And so in G1, the cell has to decide whether or not 133 00:08:23,910 --> 00:08:27,010 to enter into the cell cycle. 134 00:08:27,010 --> 00:08:30,540 So some things to consider here, the one that I mentioned 135 00:08:30,540 --> 00:08:33,135 before is whether or not there are growth signals present. 136 00:08:38,230 --> 00:08:42,440 OK, so for a metazoan cell, it's important 137 00:08:42,440 --> 00:08:46,460 that the cell doesn't just divide without any regard 138 00:08:46,460 --> 00:08:48,830 to what's going on in the surroundings. 139 00:08:48,830 --> 00:08:51,350 There needs to be a communication between cells 140 00:08:51,350 --> 00:08:53,930 such that there's the proper balance of cell 141 00:08:53,930 --> 00:08:58,340 division in a tissue for specific cell types. 142 00:08:58,340 --> 00:09:03,080 OK, so this G1 phase, this is when 143 00:09:03,080 --> 00:09:05,720 the cell-- if the cell goes from G1 to S, 144 00:09:05,720 --> 00:09:09,140 this is when the cell commits to the cell cycle. 145 00:09:09,140 --> 00:09:12,980 So G1 to S, this is the time when 146 00:09:12,980 --> 00:09:17,225 the cell commits to going through the entire cell cycle. 147 00:09:21,360 --> 00:09:25,460 So if the cell passes this G1 to S transition, 148 00:09:25,460 --> 00:09:28,820 then the cell has committed to going through the entire cell 149 00:09:28,820 --> 00:09:30,800 cycle. 150 00:09:30,800 --> 00:09:35,270 OK, the other phase, this G2 phase, 151 00:09:35,270 --> 00:09:39,650 ensures that this type of quality control that 152 00:09:39,650 --> 00:09:42,890 happens, it has to ensure that the DNA is replicated 153 00:09:42,890 --> 00:09:46,700 before the cell moves on to mitosis. 154 00:09:46,700 --> 00:09:52,850 So you can think of G2 as a phase where there's 155 00:09:52,850 --> 00:09:56,420 a quality control mechanism and the cell 156 00:09:56,420 --> 00:09:59,570 cares about whether or not its DNA is replicated or not. 157 00:10:06,400 --> 00:10:09,320 OK, so now I want to tell you basically 158 00:10:09,320 --> 00:10:16,040 the answer as to how this system works in a eukaryotic cell. 159 00:10:16,040 --> 00:10:20,000 And this system requires a level of control. 160 00:10:20,000 --> 00:10:22,010 And it requires a control system. 161 00:10:26,490 --> 00:10:28,190 And what this control system does 162 00:10:28,190 --> 00:10:30,860 is to ensure that these different events that 163 00:10:30,860 --> 00:10:34,730 happen during a cell cycle occur in the right order. 164 00:10:34,730 --> 00:10:39,395 OK, so this control system is going to ensure proper order. 165 00:10:48,980 --> 00:10:51,800 And there are two main components 166 00:10:51,800 --> 00:10:54,930 to this control system. 167 00:10:54,930 --> 00:11:01,200 The first is called cyclin dependent kinase, or CDK. 168 00:11:06,480 --> 00:11:10,050 And so cyclin dependent kinase is a kinase, 169 00:11:10,050 --> 00:11:14,570 so it can post-translationally modify other proteins 170 00:11:14,570 --> 00:11:17,370 by adding a phosphate group to them. 171 00:11:17,370 --> 00:11:20,510 And so it's through that mechanism 172 00:11:20,510 --> 00:11:25,460 that cyclin dependent kinase can modify events and control when 173 00:11:25,460 --> 00:11:27,800 they happen in the cell cycle. 174 00:11:27,800 --> 00:11:31,940 OK, and the other key component of the system 175 00:11:31,940 --> 00:11:34,280 is a protein called cyclin. 176 00:11:34,280 --> 00:11:41,130 And what cyclin is is it's the regulatory subunit of the CDK. 177 00:11:41,130 --> 00:11:45,240 So this is the regulatory subunit of CDK. 178 00:11:51,300 --> 00:11:54,910 And so without the cyclin, the cyclin dependent kinase 179 00:11:54,910 --> 00:11:56,280 is inactive, OK? 180 00:11:56,280 --> 00:12:01,860 So the CDK needs the cyclin to have activity. 181 00:12:01,860 --> 00:12:08,190 So cyclins increase the activity or activate CDK. 182 00:12:12,730 --> 00:12:16,180 OK, but there are different flavors of cyclins. 183 00:12:16,180 --> 00:12:19,440 There are actually many different cyclins, 184 00:12:19,440 --> 00:12:21,660 at least four classes of cyclins. 185 00:12:21,660 --> 00:12:25,020 And these cyclins appear at different phases of the cell 186 00:12:25,020 --> 00:12:26,700 cycle and then go away. 187 00:12:26,700 --> 00:12:29,220 OK, so the cyclins oscillate that's why they're called 188 00:12:29,220 --> 00:12:32,470 cyclins, because they come on and off. 189 00:12:32,470 --> 00:12:35,430 And depending on which cyclin is present 190 00:12:35,430 --> 00:12:39,990 determines what CDK is going to phosphorylate, OK? 191 00:12:39,990 --> 00:12:46,020 So these cyclins also determine substrate specificity 192 00:12:46,020 --> 00:12:47,520 of the kinase. 193 00:12:47,520 --> 00:12:55,230 So which cyclin determines what protein the CDK phosphorylates. 194 00:13:01,840 --> 00:13:05,110 So I've outlined three classes of cyclins here. 195 00:13:05,110 --> 00:13:10,060 Here is a G1S cyclin in complex of CDK. 196 00:13:10,060 --> 00:13:14,500 And then here's an S cyclin complex with CDK in red. 197 00:13:14,500 --> 00:13:16,630 And so what do you think the S cyclin 198 00:13:16,630 --> 00:13:21,730 CDK is going to phosphorylate, what kind of protein? 199 00:13:21,730 --> 00:13:25,060 Anyone have a guess? 200 00:13:25,060 --> 00:13:26,510 Miles-- 201 00:13:26,510 --> 00:13:27,965 AUDIENCE: Helicase. 202 00:13:27,965 --> 00:13:30,430 ADAM MARTIN: Yes, you're actually exactly right. 203 00:13:30,430 --> 00:13:33,130 It's going to phosphorylate and activate 204 00:13:33,130 --> 00:13:36,070 things that are involved in DNA replication. 205 00:13:36,070 --> 00:13:37,390 And Miles is right. 206 00:13:37,390 --> 00:13:40,210 Helicase is one of the proteins that gets phosphorylated 207 00:13:40,210 --> 00:13:42,820 by S cyclin CDK. 208 00:13:42,820 --> 00:13:48,100 And then similarly, M cyclin CDK, which appears here 209 00:13:48,100 --> 00:13:52,120 in blue during mitosis, M cyclin CDK 210 00:13:52,120 --> 00:13:54,310 is going to phosphorylate proteins 211 00:13:54,310 --> 00:13:56,680 that are involved in forming the mitotic spindle 212 00:13:56,680 --> 00:14:00,610 so that it induces cell cycle events that happen specifically 213 00:14:00,610 --> 00:14:02,500 during mitosis. 214 00:14:02,500 --> 00:14:06,580 OK, so you all see it depends which cyclin is present 215 00:14:06,580 --> 00:14:10,060 that determines what cell cycle events are happening at a given 216 00:14:10,060 --> 00:14:11,080 time. 217 00:14:11,080 --> 00:14:14,320 And therefore, it's important that we understand 218 00:14:14,320 --> 00:14:18,250 how these cyclins appear at distinct cell cycle phases 219 00:14:18,250 --> 00:14:21,040 and whether or not that's the mechanism for the oscillation. 220 00:14:21,040 --> 00:14:21,780 Yes, miles-- 221 00:14:21,780 --> 00:14:25,508 AUDIENCE: I have a question about [INAUDIBLE] question 222 00:14:25,508 --> 00:14:28,703 about mitosis [INAUDIBLE]. 223 00:14:28,703 --> 00:14:33,970 So I know that microtubules make sure the chromosomes separate 224 00:14:33,970 --> 00:14:35,140 from the cell. 225 00:14:35,140 --> 00:14:38,305 How does a cell regulate having half 226 00:14:38,305 --> 00:14:43,274 of the organelles on each side [INAUDIBLE] split [INAUDIBLE].. 227 00:14:43,274 --> 00:14:45,940 ADAM MARTIN: Some organisms employee motors 228 00:14:45,940 --> 00:14:48,100 such that organelles are physically sort 229 00:14:48,100 --> 00:14:50,590 of put in daughter cells. 230 00:14:50,590 --> 00:14:53,100 But I think often it's just random, right? 231 00:14:53,100 --> 00:14:55,600 If you dissolve the organelle and it 232 00:14:55,600 --> 00:14:58,060 becomes kind of a bunch of different vesicles, 233 00:14:58,060 --> 00:15:01,720 then if you just split in half, there's a high probability 234 00:15:01,720 --> 00:15:07,720 that each daughter cell will get parts of the organelle, OK? 235 00:15:07,720 --> 00:15:10,030 So organelles can change their morphology 236 00:15:10,030 --> 00:15:13,000 during the division process in such a way 237 00:15:13,000 --> 00:15:16,030 that they're able to be inherited by both daughter 238 00:15:16,030 --> 00:15:17,670 cells. 239 00:15:17,670 --> 00:15:21,250 OK, that's an excellent question. 240 00:15:21,250 --> 00:15:24,820 So it's the cyclins that really determine what's happening. 241 00:15:24,820 --> 00:15:26,920 And I just wanted to point out here 242 00:15:26,920 --> 00:15:32,030 that one of the main transcriptional targets of RTK 243 00:15:32,030 --> 00:15:34,690 signaling is this G1 cyclin, OK? 244 00:15:34,690 --> 00:15:37,750 So it's these signaling pathways that 245 00:15:37,750 --> 00:15:40,330 lead to the increase in G1 cyclin 246 00:15:40,330 --> 00:15:43,540 that start the cell on this process of entering 247 00:15:43,540 --> 00:15:45,290 into the cell cycle, OK? 248 00:15:50,950 --> 00:15:55,390 So you get these cyclins getting synthesized. 249 00:15:55,390 --> 00:15:58,090 And the cyclins appear in a defined order. 250 00:15:58,090 --> 00:16:05,290 OK, so there are different cyclins, 251 00:16:05,290 --> 00:16:10,000 but they appear relative to each other in a stereotypical order. 252 00:16:10,000 --> 00:16:14,440 So they appear in order. 253 00:16:14,440 --> 00:16:16,150 And it's that order of the cyclin 254 00:16:16,150 --> 00:16:18,790 that defines which cell cycle events happen 255 00:16:18,790 --> 00:16:20,590 at what time in a cell. 256 00:16:24,000 --> 00:16:27,220 OK, now I want to tell you a little bit about how 257 00:16:27,220 --> 00:16:31,060 the machinery that's involved in the control of the cell cycle 258 00:16:31,060 --> 00:16:32,710 was discovered. 259 00:16:32,710 --> 00:16:35,175 And I'm going to start by telling you 260 00:16:35,175 --> 00:16:36,550 a little bit about budding yeast. 261 00:16:40,060 --> 00:16:42,250 And by showing you how this was discovered, 262 00:16:42,250 --> 00:16:46,120 it will give you a sense as to how this system controls 263 00:16:46,120 --> 00:16:48,400 the cell division cycle. 264 00:16:48,400 --> 00:16:50,800 You'll recall that budding yeast can 265 00:16:50,800 --> 00:16:54,040 exist as a haploid cell in addition 266 00:16:54,040 --> 00:16:57,700 to existing as a diploid cell. 267 00:16:57,700 --> 00:17:00,070 So there's a haploid/diploid life cycle. 268 00:17:04,349 --> 00:17:07,390 Also, one nice feature of budding yeast 269 00:17:07,390 --> 00:17:10,569 for this particular question is that you 270 00:17:10,569 --> 00:17:13,869 can infer the cell cycle morphology of the yeast 271 00:17:13,869 --> 00:17:17,510 just by looking at its morphology. 272 00:17:17,510 --> 00:17:21,339 So budding yeast divides by budding. 273 00:17:21,339 --> 00:17:23,349 And the size of the bud indicates 274 00:17:23,349 --> 00:17:27,790 what cell cycle phase the cell is in, OK? 275 00:17:27,790 --> 00:17:36,760 So you can infer cell cycle phase 276 00:17:36,760 --> 00:17:38,590 by morphology of the yeast cell. 277 00:17:45,580 --> 00:17:50,170 So for example, if we look up here, 278 00:17:50,170 --> 00:17:54,190 here's an unbudded yeast cell that's probably in G1. 279 00:17:54,190 --> 00:17:56,830 Here's a yeast cell with a little teeny bud on it. 280 00:17:56,830 --> 00:17:59,050 That's probably an S phase. 281 00:17:59,050 --> 00:18:03,850 Next to it over here is one that's a slightly bigger bud. 282 00:18:03,850 --> 00:18:05,800 And here's one with an even bigger bud. 283 00:18:05,800 --> 00:18:07,720 And that one might be in G2 phase. 284 00:18:07,720 --> 00:18:10,810 OK, so because this bud grows in size 285 00:18:10,810 --> 00:18:12,910 over the course of the cell cycle, 286 00:18:12,910 --> 00:18:15,070 you can just look at a yeast cell and infer 287 00:18:15,070 --> 00:18:19,120 what the cell cycle phase is. 288 00:18:19,120 --> 00:18:22,690 OK, so I'm going to tell you about a genetic screen that 289 00:18:22,690 --> 00:18:28,660 was done to look for mutants that were defective in the cell 290 00:18:28,660 --> 00:18:30,820 division cycle. 291 00:18:30,820 --> 00:18:32,650 And these are known as cell division 292 00:18:32,650 --> 00:18:36,355 cycle, or CDC, mutants. 293 00:18:41,380 --> 00:18:44,500 Now, what type of yeast cell, haploid of diploid, might 294 00:18:44,500 --> 00:18:47,350 you want to screen mutants with? 295 00:18:50,100 --> 00:18:55,170 What would be the advantages of either one or the other? 296 00:18:55,170 --> 00:18:55,770 Yeah, Natalie? 297 00:18:55,770 --> 00:18:58,180 AUDIENCE: Would you do haploid, because if it's 298 00:18:58,180 --> 00:19:01,072 a recessive mutation [INAUDIBLE] expressed? 299 00:19:01,072 --> 00:19:03,330 ADAM MARTIN: Yes, so what Natalie 300 00:19:03,330 --> 00:19:06,870 suggested is to start with the haploid mutants 301 00:19:06,870 --> 00:19:09,990 because there's only one copy of each gene such 302 00:19:09,990 --> 00:19:13,290 that if you hit it, now you no longer have a functional 303 00:19:13,290 --> 00:19:14,640 copy of that gene. 304 00:19:14,640 --> 00:19:18,060 If you started with a diploid cell, 305 00:19:18,060 --> 00:19:20,040 you'd have two copies of the gene. 306 00:19:20,040 --> 00:19:23,610 And you'd have to have two mutations both happening 307 00:19:23,610 --> 00:19:26,410 in the same gene, which would be a rare event. 308 00:19:26,410 --> 00:19:30,390 OK, so it's better to start with a haploid in this case. 309 00:19:30,390 --> 00:19:32,580 Now, what's the problem if you mutate a gene that's 310 00:19:32,580 --> 00:19:34,690 involved in the cell cycle? 311 00:19:34,690 --> 00:19:36,660 What's going to be the phenotype, 312 00:19:36,660 --> 00:19:39,786 the immediate visible phenotype? 313 00:19:39,786 --> 00:19:41,830 Is it going to be alive or dead? 314 00:19:41,830 --> 00:19:42,330 Carmen-- 315 00:19:42,330 --> 00:19:43,050 AUDIENCE: Dead. 316 00:19:43,050 --> 00:19:44,960 ADAM MARTIN: It's going to be dead, right? 317 00:19:44,960 --> 00:19:48,690 And it's hard to work with an organism that's dead. 318 00:19:48,690 --> 00:19:53,430 OK, so what was done is to look for a particular type of mutant 319 00:19:53,430 --> 00:19:55,860 which is known as a temperature sensitive mutant. 320 00:20:04,740 --> 00:20:07,050 And a temperature sensitive mutant 321 00:20:07,050 --> 00:20:10,410 is a mutant where the cell or organism 322 00:20:10,410 --> 00:20:13,050 is alive and well at one temperature 323 00:20:13,050 --> 00:20:16,230 but dead at another temperature, OK? 324 00:20:16,230 --> 00:20:21,090 And so the screen basically involved taking yeast. 325 00:20:21,090 --> 00:20:23,880 Here's yeast growing in a test tube. 326 00:20:23,880 --> 00:20:26,805 And this is now haploid yeast. 327 00:20:29,730 --> 00:20:32,330 And you can treat that yeast with a mutagen. 328 00:20:32,330 --> 00:20:34,410 It doesn't matter what, just something that 329 00:20:34,410 --> 00:20:38,520 will induce mutations at a high rate in these yeast cells. 330 00:20:38,520 --> 00:20:40,620 And then you can take these cells 331 00:20:40,620 --> 00:20:45,150 and plate them on media where individual cells will 332 00:20:45,150 --> 00:20:46,095 grow into colonies. 333 00:20:50,330 --> 00:20:56,020 OK, and if you grow it at 22 degrees C for yeast, this 334 00:20:56,020 --> 00:20:58,750 is the most moderate temperature you can choose. 335 00:20:58,750 --> 00:21:01,723 So this is what's known as the permissive temperature. 336 00:21:05,110 --> 00:21:10,020 OK, but you can also take this plate of used colonies 337 00:21:10,020 --> 00:21:14,677 and duplicate it and grow it at another temperature. 338 00:21:14,677 --> 00:21:16,260 And you might get something that looks 339 00:21:16,260 --> 00:21:21,000 like this, where you see this colony grew at 22 degrees, 340 00:21:21,000 --> 00:21:24,900 but at 37 degrees C it did not grow. 341 00:21:24,900 --> 00:21:27,180 And that would suggest that, then, this has 342 00:21:27,180 --> 00:21:30,650 a temperature sensitive mutant. 343 00:21:30,650 --> 00:21:32,970 And this temperature of 37 degrees 344 00:21:32,970 --> 00:21:35,214 is known as the restrictive temperature. 345 00:21:40,430 --> 00:21:44,130 OK, so that would identify a temperature sensitive mutant. 346 00:21:44,130 --> 00:21:46,380 Now, is every temperature sensitive mutant 347 00:21:46,380 --> 00:21:50,790 that you identify, is that going to be a cell division cycle 348 00:21:50,790 --> 00:21:53,460 mutant? 349 00:21:53,460 --> 00:21:55,410 Miles, you're shaking your head no. 350 00:21:55,410 --> 00:21:56,070 Why is that? 351 00:21:56,070 --> 00:21:57,210 Can you explain your logic? 352 00:21:57,210 --> 00:22:02,060 AUDIENCE: So there could be a couple different proteins 353 00:22:02,060 --> 00:22:04,360 [INAUDIBLE] there's too many mechanisms 354 00:22:04,360 --> 00:22:08,050 in the cell that could be dependent on temperature 355 00:22:08,050 --> 00:22:11,520 to narrow down to just [INAUDIBLE] mutant cell cycle. 356 00:22:11,520 --> 00:22:15,500 For example, if a phytoprotein mutant organism 357 00:22:15,500 --> 00:22:17,590 would mutate [INAUDIBLE] temperature sensitive, 358 00:22:17,590 --> 00:22:20,084 without that protein it would die also. 359 00:22:20,084 --> 00:22:21,140 ADAM MARTIN: Exactly. 360 00:22:21,140 --> 00:22:26,340 So Miles is suggesting that if you just mutated 361 00:22:26,340 --> 00:22:31,800 any old gene that was involved in viability for this yeast, 362 00:22:31,800 --> 00:22:36,690 and it unfolded at 37 degrees because you sort of made 363 00:22:36,690 --> 00:22:39,250 a mutation that made it unstable, 364 00:22:39,250 --> 00:22:43,210 then you would identify that as a temperature sensitive mutant. 365 00:22:43,210 --> 00:22:45,240 So what would be a good criteria, I guess, 366 00:22:45,240 --> 00:22:48,840 that we could use to select just the mutants that are affecting 367 00:22:48,840 --> 00:22:51,480 the cell division cycle? 368 00:22:51,480 --> 00:22:55,240 Might there be a way for us to do that? 369 00:22:55,240 --> 00:22:57,930 I guess I'm asking, can we narrow down the phenotype, 370 00:22:57,930 --> 00:22:58,500 right? 371 00:22:58,500 --> 00:23:00,480 Temperature sensitivity could be-- 372 00:23:00,480 --> 00:23:02,790 by affecting any process in yeast, 373 00:23:02,790 --> 00:23:05,200 is there a way we can gear it towards the cell division 374 00:23:05,200 --> 00:23:05,700 cycle? 375 00:23:09,470 --> 00:23:10,327 Diana-- 376 00:23:10,327 --> 00:23:12,820 AUDIENCE: Maybe [INAUDIBLE] specific phase of the cell 377 00:23:12,820 --> 00:23:16,175 cycle, you could look at the morphology of it. 378 00:23:16,175 --> 00:23:19,080 And if all of them stop at the same phase, 379 00:23:19,080 --> 00:23:21,383 you might assume that you [INAUDIBLE].. 380 00:23:21,383 --> 00:23:22,800 ADAM MARTIN: All right, very good. 381 00:23:22,800 --> 00:23:27,200 So Diana is suggesting is that we looked for a phenotype. 382 00:23:27,200 --> 00:23:28,950 And she's guessed that the phenotype, 383 00:23:28,950 --> 00:23:34,430 if this gene is involved in sort of mediating a change from one 384 00:23:34,430 --> 00:23:37,250 cell cycle to phase to another, that if you 385 00:23:37,250 --> 00:23:41,360 mutate that, you'd have yeast that's all stuck in one phase, 386 00:23:41,360 --> 00:23:42,170 OK? 387 00:23:42,170 --> 00:23:47,450 And that's indeed the phenotype that was screened for. 388 00:23:47,450 --> 00:23:51,650 OK, and so if you just take a random population of yeast 389 00:23:51,650 --> 00:23:55,100 that's dividing, you'll see cells 390 00:23:55,100 --> 00:23:59,050 that are unbudded, small budded, slightly bigger budded. 391 00:23:59,050 --> 00:24:02,720 And if you count the number of cells, what you'll see 392 00:24:02,720 --> 00:24:05,750 is that most of your cells are unbudded. 393 00:24:05,750 --> 00:24:07,370 Some are small budded. 394 00:24:07,370 --> 00:24:10,850 And a larger percentage are large budded. 395 00:24:10,850 --> 00:24:14,480 And this just reflects the relative amount of time 396 00:24:14,480 --> 00:24:18,300 that yeast is in each of these phases of the cell cycle. 397 00:24:18,300 --> 00:24:20,990 So yeast spends most of its time in G1. 398 00:24:20,990 --> 00:24:24,170 Therefore, if you look at a random population of yeast, 399 00:24:24,170 --> 00:24:28,350 you'll see most of the cells will be in the unbudded state. 400 00:24:28,350 --> 00:24:31,550 OK, so this is for wild-type normal yeast. 401 00:24:34,430 --> 00:24:38,140 Now, what was identified is a cell division cycle mutant, 402 00:24:38,140 --> 00:24:42,790 CDC 28, which at the restrictive temperature 403 00:24:42,790 --> 00:24:46,450 causes a train wreck at a specific phase of the cell 404 00:24:46,450 --> 00:24:48,980 cycle. 405 00:24:48,980 --> 00:24:53,620 So all of the cells now are stuck in the unbudded state. 406 00:24:56,290 --> 00:25:00,400 And so this suggests that these cells, when 407 00:25:00,400 --> 00:25:03,100 they are shifted to the restrictive temperature, 408 00:25:03,100 --> 00:25:05,530 are still able to move through the cell cycle. 409 00:25:05,530 --> 00:25:09,650 But once they get to this phase, they get stuck. 410 00:25:09,650 --> 00:25:20,360 OK, so here there is a cell cycle arrest at G1. 411 00:25:20,360 --> 00:25:24,870 And they confirmed it was G1 by measuring the DNA content. 412 00:25:24,870 --> 00:25:26,660 And by measuring the DNA content, 413 00:25:26,660 --> 00:25:33,190 they were able to show that these cells did not 414 00:25:33,190 --> 00:25:34,730 duplicate their DNA. 415 00:25:34,730 --> 00:25:37,120 So they didn't even start to undergo S phase. 416 00:25:37,120 --> 00:25:39,140 They were stuck in G1, OK? 417 00:25:43,380 --> 00:25:50,890 OK, so that suggests that the CDC 28 gene is required 418 00:25:50,890 --> 00:25:53,440 for cells to go from G1 to Sl. 419 00:25:53,440 --> 00:25:56,680 OK, so the wild-type CDC 28 gene is 420 00:25:56,680 --> 00:26:01,660 required for this transition from G1 into the S phase. 421 00:26:01,660 --> 00:26:06,370 And it turns out that this yeast CDC 28 gene is the one yeast 422 00:26:06,370 --> 00:26:10,210 cyclin dependent kinase, OK? 423 00:26:10,210 --> 00:26:14,620 So this was sort of the defining mutant 424 00:26:14,620 --> 00:26:16,570 for cyclin dependent kinase. 425 00:26:16,570 --> 00:26:18,400 And you'll recall earlier in the semester 426 00:26:18,400 --> 00:26:21,160 when we talked about molecular biology 427 00:26:21,160 --> 00:26:25,030 that we talked about work done by Paul Nurse who 428 00:26:25,030 --> 00:26:27,940 used functional complementation to clone 429 00:26:27,940 --> 00:26:30,520 the human cyclin dependent kinase 430 00:26:30,520 --> 00:26:35,050 by transforming DNA into a different yeast, fission yeast. 431 00:26:35,050 --> 00:26:38,080 But again, that rescued the cell cycle arrest. 432 00:26:38,080 --> 00:26:40,810 And that's how the human cyclin dependent kinase 433 00:26:40,810 --> 00:26:43,450 was discovered. 434 00:26:43,450 --> 00:26:45,040 And I just wanted to point out here 435 00:26:45,040 --> 00:26:48,400 that the work I'm telling you about 436 00:26:48,400 --> 00:26:51,520 was awarded the Nobel Prize in physiology and medicine 437 00:26:51,520 --> 00:26:53,260 in 2001. 438 00:26:53,260 --> 00:26:56,380 And it was awarded to Leland Hartwell, Tim Hunt, 439 00:26:56,380 --> 00:26:57,940 and Sir Paul Nurse. 440 00:26:57,940 --> 00:26:59,920 Leland Hartwell did the screen that I just 441 00:26:59,920 --> 00:27:03,100 told you about there and identified CDC 28. 442 00:27:03,100 --> 00:27:06,880 And we already talked about Paul Nurse earlier in the semester. 443 00:27:06,880 --> 00:27:11,710 Tim Hunt worked on clams and sea urchins and identified cyclin. 444 00:27:11,710 --> 00:27:13,900 So this was the work that identified 445 00:27:13,900 --> 00:27:16,690 the regulatory machinery of the cell cycle. 446 00:27:16,690 --> 00:27:19,480 And they sort of showed that this 447 00:27:19,480 --> 00:27:21,820 worked in a number of different organisms. 448 00:27:21,820 --> 00:27:23,830 And they showed that it was evolutionarily 449 00:27:23,830 --> 00:27:26,760 conserved from yeast all the way to humans. 450 00:27:26,760 --> 00:27:28,990 OK, so this is a conserved mechanism. 451 00:27:33,470 --> 00:27:38,640 OK, so there's a mechanism that actively governs 452 00:27:38,640 --> 00:27:41,610 the transition from G1 to S. And I'll 453 00:27:41,610 --> 00:27:46,710 point out this transition is known as start in yeast. 454 00:27:46,710 --> 00:27:49,215 And it's called the restriction point in mammalian cells. 455 00:27:49,215 --> 00:27:51,795 It's kind of the point of no return in the cell cycle. 456 00:27:54,540 --> 00:27:56,970 But the cell cycle doesn't just blindly 457 00:27:56,970 --> 00:27:59,220 charge through the rest of the way. 458 00:27:59,220 --> 00:28:01,560 And there are certain quality control mechanisms 459 00:28:01,560 --> 00:28:04,140 that are in place to ensure that things happen 460 00:28:04,140 --> 00:28:08,340 in the proper order and that the quality of events happening 461 00:28:08,340 --> 00:28:12,220 is good before the cell moves on to the next stage. 462 00:28:12,220 --> 00:28:17,070 And so I'm going to define a concept called a checkpoint. 463 00:28:17,070 --> 00:28:20,420 And the checkpoint is a type of quality control mechanism. 464 00:28:24,450 --> 00:28:27,120 And checkpoints operate in the cell cycle 465 00:28:27,120 --> 00:28:34,830 to ensure that one event doesn't occur till the preceding 466 00:28:34,830 --> 00:28:39,780 event happens correctly, OK? 467 00:28:39,780 --> 00:28:47,560 So this ensures proper order of events 468 00:28:47,560 --> 00:28:50,140 and ensures that events happen correctly 469 00:28:50,140 --> 00:28:54,670 before the next subsequent event has to occur. 470 00:28:54,670 --> 00:28:57,520 OK, so one example of this is, if you just 471 00:28:57,520 --> 00:29:01,330 consider S phase and M phase, DNA replication 472 00:29:01,330 --> 00:29:05,350 has to finish before the cell starts segregating chromosomes. 473 00:29:05,350 --> 00:29:09,310 Otherwise there's going to be catastrophic consequences, 474 00:29:09,310 --> 00:29:13,510 such as possibly creating a cancer cell, OK? 475 00:29:13,510 --> 00:29:18,310 So one example of a checkpoint is called the DNA damage 476 00:29:18,310 --> 00:29:19,010 checkpoint. 477 00:29:25,870 --> 00:29:29,020 And what the DNA damage checkpoint does 478 00:29:29,020 --> 00:29:35,340 is it looks to see if there's DNA damage 479 00:29:35,340 --> 00:29:37,530 or if the DNA is still replicating. 480 00:29:41,860 --> 00:29:45,460 And if either of these cases is present in a cell, 481 00:29:45,460 --> 00:29:46,555 then it sends a signal. 482 00:29:52,110 --> 00:29:57,120 And that signal, in order to influence the cell division 483 00:29:57,120 --> 00:30:02,640 cycle, has to interface with the cyclin CDK control machinery, 484 00:30:02,640 --> 00:30:03,600 OK? 485 00:30:03,600 --> 00:30:06,800 So this signal will then inhibit cyclin CDK. 486 00:30:09,770 --> 00:30:16,920 And cyclin CDK governs two major transitions in the cell cycle. 487 00:30:16,920 --> 00:30:19,785 So there are two major what I'll call transition points. 488 00:30:24,300 --> 00:30:28,210 There's the G1 to S, which I just outlined over there, 489 00:30:28,210 --> 00:30:29,400 which is called start. 490 00:30:29,400 --> 00:30:35,790 So there's G1 to S. But there's also G2 to M, OK? 491 00:30:35,790 --> 00:30:39,030 So these, basically, the transition out of the gap 492 00:30:39,030 --> 00:30:41,820 phases, those are the key transition points 493 00:30:41,820 --> 00:30:44,280 that can be regulated by the cell 494 00:30:44,280 --> 00:30:48,720 to either slow things down to halt the transition 495 00:30:48,720 --> 00:30:52,590 or just go right through, OK? 496 00:30:52,590 --> 00:30:55,080 So let me tell you about an experiment that 497 00:30:55,080 --> 00:31:00,840 defined the functionality of the DNA damage checkpoint. 498 00:31:00,840 --> 00:31:06,475 And I'm going to tell you about work done by Weinert and Leland 499 00:31:06,475 --> 00:31:06,975 Hartwell. 500 00:31:09,900 --> 00:31:12,930 And it was published in 1988. 501 00:31:12,930 --> 00:31:17,100 And they were interested in what the nature and the function 502 00:31:17,100 --> 00:31:19,710 of these checkpoints was. 503 00:31:19,710 --> 00:31:22,800 And so you can take budding yeast. 504 00:31:22,800 --> 00:31:26,260 And you can damage its DNA by irradiating the cells 505 00:31:26,260 --> 00:31:26,760 with X-rays. 506 00:31:30,600 --> 00:31:33,360 And if you irradiate the cell with X-rays 507 00:31:33,360 --> 00:31:38,700 in a wild-type normal yeast, so in the normal yeast that's 508 00:31:38,700 --> 00:31:43,710 not mutant, then this damages the DNA. 509 00:31:43,710 --> 00:31:46,580 And the cell stays in G2. 510 00:31:46,580 --> 00:31:47,975 OK, so it stays in G2. 511 00:31:50,890 --> 00:31:52,170 OK, and there's a delay. 512 00:31:52,170 --> 00:31:54,750 OK, so here what I'm drawing is a G2 delay. 513 00:31:57,670 --> 00:32:02,070 So the cell spends an abnormally longer time in G2 514 00:32:02,070 --> 00:32:04,875 than it normally would if you didn't damage its DNA. 515 00:32:07,410 --> 00:32:11,910 All right, and then over time it will continue in the cell cycle 516 00:32:11,910 --> 00:32:14,700 and enter into the next cell cycle. 517 00:32:14,700 --> 00:32:20,970 And what's interesting about this is that these cells live. 518 00:32:20,970 --> 00:32:24,360 OK, so one interpretation from this result 519 00:32:24,360 --> 00:32:27,390 is you damage the cell's DNA. 520 00:32:27,390 --> 00:32:29,820 It delayed the cell cycle in G2. 521 00:32:29,820 --> 00:32:33,450 So it didn't rush right into chromosome segregation. 522 00:32:33,450 --> 00:32:36,960 And that allowed the cell time to repair its DNA. 523 00:32:36,960 --> 00:32:41,580 And that enabled the cell daughters to live, OK? 524 00:32:41,580 --> 00:32:44,050 So that's an interpretation. 525 00:32:44,050 --> 00:32:48,270 Now, part of the evidence for that interpretation 526 00:32:48,270 --> 00:32:52,560 is that Hartwell and Weinert discovered a mutant called 527 00:32:52,560 --> 00:32:56,190 RAD 9, so the RAD 9 mutant. 528 00:32:56,190 --> 00:32:59,140 And RAD 9 stands for radiation sensitive. 529 00:32:59,140 --> 00:33:01,530 This is a radiation sensitive mutant. 530 00:33:01,530 --> 00:33:04,590 And this particular radiation sensitive mutant 531 00:33:04,590 --> 00:33:06,750 disrupted the delay. 532 00:33:06,750 --> 00:33:09,930 So it disrupted the checkpoint here. 533 00:33:09,930 --> 00:33:13,320 So what happens in a RAD 9 mutant is, 534 00:33:13,320 --> 00:33:15,300 again, you irradiate cells with X-rays. 535 00:33:18,630 --> 00:33:22,440 The cell goes from S phase to G2. 536 00:33:22,440 --> 00:33:28,770 But this time, there's no delay, so it goes from G2, 537 00:33:28,770 --> 00:33:31,830 the poor yeast charges unsuspectingly 538 00:33:31,830 --> 00:33:36,600 into mitosis with damaged DNA and divides. 539 00:33:36,600 --> 00:33:38,880 But in this case, there's a high level 540 00:33:38,880 --> 00:33:42,220 of death in the resulting progeny. 541 00:33:42,220 --> 00:33:43,320 So here you have death. 542 00:33:46,290 --> 00:33:49,570 OK, so RAD 9, then, is a gene that 543 00:33:49,570 --> 00:33:53,710 is involved in promoting the cell cycle delay such 544 00:33:53,710 --> 00:33:57,200 that the yeast cell has time to repair its DNA. 545 00:33:57,200 --> 00:33:58,990 And if you remove that delay-- 546 00:33:58,990 --> 00:34:01,045 so here there's no delay. 547 00:34:04,720 --> 00:34:08,810 If you disrupt this delay, which defines the checkpoint process, 548 00:34:08,810 --> 00:34:09,310 right? 549 00:34:09,310 --> 00:34:13,150 The checkpoint is a process whereby if there's DNA damage, 550 00:34:13,150 --> 00:34:15,850 you delay the cell cycle such that the cell has 551 00:34:15,850 --> 00:34:17,770 time to repair it. 552 00:34:17,770 --> 00:34:21,489 If you don't have that, it has bad consequences 553 00:34:21,489 --> 00:34:25,210 for the cell and results in your cells undergoing 554 00:34:25,210 --> 00:34:29,345 premature mitosis before they've had a chance to repair the DNA. 555 00:34:35,262 --> 00:34:36,000 Let's see. 556 00:34:36,000 --> 00:34:39,600 I'm going to use this one here. 557 00:34:39,600 --> 00:34:42,030 All right, one thing you might be wondering 558 00:34:42,030 --> 00:34:47,489 is what causes these cyclin proteins to oscillate. 559 00:34:47,489 --> 00:34:49,560 And so the last point I want to make 560 00:34:49,560 --> 00:34:52,650 is I want to tell you about the mechanism that allows 561 00:34:52,650 --> 00:34:55,230 these protein oscillations. 562 00:34:55,230 --> 00:34:57,630 And it involves a mechanism that's 563 00:34:57,630 --> 00:35:00,540 going to be new for you, at least 564 00:35:00,540 --> 00:35:02,740 from the context of this class. 565 00:35:02,740 --> 00:35:05,740 It's a mechanism that's regulated proteolysis. 566 00:35:11,790 --> 00:35:16,380 OK, so there's a regulated degradation of these cyclin 567 00:35:16,380 --> 00:35:20,880 proteins that allows them to go up and then down, OK? 568 00:35:20,880 --> 00:35:25,980 So if we consider just one part of the cell cycle, 569 00:35:25,980 --> 00:35:30,940 well, if I plot the concentration of cyclin 570 00:35:30,940 --> 00:35:34,630 and we look at M cyclin from G2 to M phase-- 571 00:35:34,630 --> 00:35:37,840 so this here is a time axis-- 572 00:35:37,840 --> 00:35:41,970 the M cyclin goes up in M phase. 573 00:35:41,970 --> 00:35:46,350 And then it drops precipitously during mitosis, specifically 574 00:35:46,350 --> 00:35:54,580 at the metaphase/anaphase OK, so this is for the mitotic cyclin. 575 00:35:54,580 --> 00:35:58,080 And I'm going to tell you that this precipitous decline 576 00:35:58,080 --> 00:36:02,690 in cyclin levels is due to regulated proteolysis. 577 00:36:07,410 --> 00:36:11,580 OK, and it involves a mechanism that you were briefly 578 00:36:11,580 --> 00:36:14,400 introduced to by Professor Imperiali, 579 00:36:14,400 --> 00:36:18,620 because it involves a small protein known as ubiquitin. 580 00:36:21,840 --> 00:36:27,180 What ubiquitin is is it's a small 76 amino acid protein. 581 00:36:27,180 --> 00:36:29,295 So it's a 76 amino acid protein. 582 00:36:31,980 --> 00:36:35,520 But this protein can get attached to other proteins. 583 00:36:35,520 --> 00:36:39,170 So it's a post-translational modification. 584 00:36:39,170 --> 00:36:43,740 OK, so ubiquitin, which I'll abbreviate UB, 585 00:36:43,740 --> 00:36:52,385 ubiquitin is attached to lysines on a target protein. 586 00:37:02,390 --> 00:37:07,070 And the attachment of ubiquitins to lysines of a protein 587 00:37:07,070 --> 00:37:09,420 has important consequences. 588 00:37:09,420 --> 00:37:11,180 And what Professor Imperiali told 589 00:37:11,180 --> 00:37:15,140 you about is when this happens in the case of protein 590 00:37:15,140 --> 00:37:16,610 misfolding. 591 00:37:16,610 --> 00:37:19,430 But this ubiquitination of proteins 592 00:37:19,430 --> 00:37:24,080 also occurs to proteins that are not denatured or misfolded. 593 00:37:24,080 --> 00:37:28,160 And it's a way of regulating protein levels in the cell. 594 00:37:28,160 --> 00:37:30,980 OK, so what happens is-- 595 00:37:33,950 --> 00:37:36,980 I'll show you a complicated diagram of what happens. 596 00:37:36,980 --> 00:37:39,650 But I'm really going to focus on this step right here. 597 00:37:39,650 --> 00:37:41,600 There's a series of steps that are 598 00:37:41,600 --> 00:37:46,730 needed to get ubiquitin to get attached to the target protein. 599 00:37:46,730 --> 00:37:49,830 I'm going to ignore pretty much all of that. 600 00:37:49,830 --> 00:37:52,580 But there is an E2 enzyme that becomes 601 00:37:52,580 --> 00:37:53,980 conjugated with ubiquitin. 602 00:37:53,980 --> 00:37:58,545 And then the ubiquitin is able to be transferred to a target 603 00:37:58,545 --> 00:37:59,045 protein. 604 00:38:02,720 --> 00:38:05,110 And rather than make this generic, 605 00:38:05,110 --> 00:38:08,463 I'll let you know the target protein is going to be cyclin. 606 00:38:08,463 --> 00:38:09,505 So we'll just say cyclin. 607 00:38:14,470 --> 00:38:18,050 And so there's an enzyme that transfers the ubiquitin 608 00:38:18,050 --> 00:38:24,710 from the E2 to the cyclin, OK? 609 00:38:24,710 --> 00:38:27,080 And it's polyubiquitinated, meaning 610 00:38:27,080 --> 00:38:31,820 there's a chain of ubiquitins added to the cyclin. 611 00:38:31,820 --> 00:38:35,900 And it's carried out by a particular type of enzyme known 612 00:38:35,900 --> 00:38:37,610 as an E3 ubiquitin ligase. 613 00:38:42,020 --> 00:38:46,250 And there are hundreds of these ubiquitin ligases in humans. 614 00:38:46,250 --> 00:38:50,180 And different E3 ubiquitin ligases 615 00:38:50,180 --> 00:38:51,920 confer different specificities. 616 00:38:51,920 --> 00:38:54,800 So they target different proteins, OK? 617 00:38:54,800 --> 00:38:57,710 So different E3's target different proteins. 618 00:39:00,230 --> 00:39:04,970 And so this is where the specificity comes from, OK? 619 00:39:04,970 --> 00:39:08,930 So when a protein in the cell, misfolded or not, 620 00:39:08,930 --> 00:39:12,620 is polyubiquitinated like this, this 621 00:39:12,620 --> 00:39:14,900 is a garbage tag on that protein, OK? 622 00:39:14,900 --> 00:39:18,080 So you can think of polyubiquitin as a garbage tag. 623 00:39:22,580 --> 00:39:25,390 And once it's polyubiquitinated, it's 624 00:39:25,390 --> 00:39:30,060 sent to the proteasome, which Professor Imperiali showed you. 625 00:39:30,060 --> 00:39:33,500 And this is the structure that degrades proteins. 626 00:39:33,500 --> 00:39:38,270 OK, so if the protein is targeted for degradation 627 00:39:38,270 --> 00:39:42,170 by putting this tag on it, it's going to be rapidly proteolyzed 628 00:39:42,170 --> 00:39:43,760 in the cytoplasm of the cell. 629 00:39:47,290 --> 00:39:50,720 All right, now I want to show you some experiments that 630 00:39:50,720 --> 00:39:55,520 provided the first evidence that this regulated proteolysis is 631 00:39:55,520 --> 00:39:59,480 what sort of causes the cyclin to oscillate, OK? 632 00:39:59,480 --> 00:40:02,720 And it's going to involve a new model organism. 633 00:40:02,720 --> 00:40:07,778 Is there anyone here that has ranidaphobia? 634 00:40:07,778 --> 00:40:09,560 OK, we all like frogs? 635 00:40:09,560 --> 00:40:15,260 OK, so this model organism is xenopus laevis, 636 00:40:15,260 --> 00:40:17,330 or the African clawed frog. 637 00:40:17,330 --> 00:40:20,060 And I want to thank my colleague at UMass Amherst, Tom 638 00:40:20,060 --> 00:40:24,620 [? Oreska, ?] who provided the slides of frogs for me. 639 00:40:24,620 --> 00:40:28,010 So what's great about these frogs, 640 00:40:28,010 --> 00:40:30,410 well, other than them being very cute, 641 00:40:30,410 --> 00:40:32,900 is that they lay a ton of eggs. 642 00:40:32,900 --> 00:40:34,760 And the eggs are huge, OK? 643 00:40:34,760 --> 00:40:37,760 So here is a mom, a mom frog. 644 00:40:37,760 --> 00:40:40,850 And then you see all these circular things all around 645 00:40:40,850 --> 00:40:42,620 the frog are the eggs. 646 00:40:42,620 --> 00:40:45,110 OK, so they're about 1 millimeter in size. 647 00:40:45,110 --> 00:40:46,190 They're huge. 648 00:40:46,190 --> 00:40:49,700 You can collect these eggs, put them in a test tube. 649 00:40:49,700 --> 00:40:51,590 And you can see all these eggs that you 650 00:40:51,590 --> 00:40:53,510 have in this test tube. 651 00:40:53,510 --> 00:40:56,390 And then you can spin the test tube. 652 00:40:56,390 --> 00:41:00,320 And by spinning the test tube in a centrifuge, 653 00:41:00,320 --> 00:41:03,680 you crush the eggs. 654 00:41:03,680 --> 00:41:06,200 And so that results in this middle layer 655 00:41:06,200 --> 00:41:10,680 here, which is cytoplasm, OK? 656 00:41:10,680 --> 00:41:14,470 And you can remove it with a syringe. 657 00:41:14,470 --> 00:41:19,190 And what you end up with is a concentrated cytoplasmic 658 00:41:19,190 --> 00:41:19,690 extract. 659 00:41:22,980 --> 00:41:25,780 So that's all cytoplasm. 660 00:41:25,780 --> 00:41:28,960 OK, so that's a lot of cytoplasm. 661 00:41:35,150 --> 00:41:40,230 OK, so for the xenopus system, this system 662 00:41:40,230 --> 00:41:43,050 allows you to get this highly concentrated-- 663 00:41:43,050 --> 00:41:44,440 because it's not diluted. 664 00:41:44,440 --> 00:41:48,150 It's the same concentration as cytoplasm almost-- 665 00:41:48,150 --> 00:41:56,700 cytoplasmic extract, which is known as xenopus egg extract. 666 00:41:56,700 --> 00:42:00,720 OK, and what's amazing about this egg extract 667 00:42:00,720 --> 00:42:03,360 is it can go through the cell cycle 668 00:42:03,360 --> 00:42:04,980 even though it's not in a cell. 669 00:42:04,980 --> 00:42:09,060 OK, so you can get this extract to essentially simulate 670 00:42:09,060 --> 00:42:10,470 the cell cycle. 671 00:42:10,470 --> 00:42:13,470 OK, so you have to mimic fertilization, 672 00:42:13,470 --> 00:42:17,460 because that's when cell divisions start to happen 673 00:42:17,460 --> 00:42:20,610 in the normal frog embryo. 674 00:42:20,610 --> 00:42:25,290 But once you do this, then you mimic the fertilization process 675 00:42:25,290 --> 00:42:27,660 by adding calcium, and then you'll 676 00:42:27,660 --> 00:42:30,640 see this extract go through the cell cycle. 677 00:42:30,640 --> 00:42:32,790 And you can see it by looking at the morphology 678 00:42:32,790 --> 00:42:35,430 of different structures in the extract. 679 00:42:35,430 --> 00:42:39,210 So this is a nucleus that has assembled an extract 680 00:42:39,210 --> 00:42:42,340 around some DNA that was added. 681 00:42:42,340 --> 00:42:46,800 OK, DNA replication can happen in this nucleus. 682 00:42:46,800 --> 00:42:49,890 Other events that happen in the interphase of the cell cycle 683 00:42:49,890 --> 00:42:52,530 also happen in this extract. 684 00:42:52,530 --> 00:42:55,830 And if you wait, then it will go into mitosis. 685 00:42:55,830 --> 00:42:58,620 And you'll start to see mitotic spindles assembling 686 00:42:58,620 --> 00:42:59,590 in the extract. 687 00:42:59,590 --> 00:43:04,170 OK, so what's important about this, this is totally in vitro. 688 00:43:04,170 --> 00:43:07,020 OK, so this is an in vitro system. 689 00:43:07,020 --> 00:43:08,660 There are no cells. 690 00:43:08,660 --> 00:43:13,620 But you're able to see the extract go through the cell 691 00:43:13,620 --> 00:43:14,370 cycle. 692 00:43:14,370 --> 00:43:19,020 OK, and if you were able to look at cyclin, like M cyclin, 693 00:43:19,020 --> 00:43:22,990 you'd see that M cyclin levels go up and then down and up 694 00:43:22,990 --> 00:43:23,490 and down. 695 00:43:23,490 --> 00:43:27,940 They oscillate just like they would in a cell. 696 00:43:27,940 --> 00:43:29,650 And this is just a diagram showing you 697 00:43:29,650 --> 00:43:34,430 that here where mitotic cyclin, M cyclin, concentration 698 00:43:34,430 --> 00:43:39,260 is in blue and CDK activity for M cyclin CDK is in purple. 699 00:43:39,260 --> 00:43:41,150 So you see it goes up. 700 00:43:41,150 --> 00:43:43,370 And then the cell enters mitosis. 701 00:43:43,370 --> 00:43:44,930 Early mitosis is in blue. 702 00:43:44,930 --> 00:43:47,280 Late mitosis is an orange. 703 00:43:47,280 --> 00:43:50,420 So you see mitosis happens when M cyclin is high, 704 00:43:50,420 --> 00:43:53,060 just like it does in a cell. 705 00:43:53,060 --> 00:43:54,500 And then it degrades. 706 00:43:54,500 --> 00:43:56,030 And then it repeats. 707 00:43:56,030 --> 00:43:58,700 OK, so this is all outside of a cell. 708 00:43:58,700 --> 00:44:00,650 But you're just looking in a test tube. 709 00:44:00,650 --> 00:44:03,960 And you can recreate the cell cycle. 710 00:44:03,960 --> 00:44:06,800 Now, this, because this is a biochemical system, 711 00:44:06,800 --> 00:44:08,570 allowed these researchers-- 712 00:44:08,570 --> 00:44:11,210 in this case, the researchers who did this experiment 713 00:44:11,210 --> 00:44:14,670 were Andrew Murray and Mark Kirchner at Harvard. 714 00:44:14,670 --> 00:44:18,270 And what they did was to test the role of various components 715 00:44:18,270 --> 00:44:19,970 in this oscillation. 716 00:44:19,970 --> 00:44:21,710 The first experiment they did was 717 00:44:21,710 --> 00:44:25,910 to RNase treat the extract to get rid of all the mRNA. 718 00:44:25,910 --> 00:44:29,720 And if you degrade all of the mRNA in this extract, 719 00:44:29,720 --> 00:44:31,580 you no longer get the cycling. 720 00:44:31,580 --> 00:44:33,680 You no longer get the cell cycle. 721 00:44:33,680 --> 00:44:38,240 So this shows you mRNA is important or necessary. 722 00:44:41,090 --> 00:44:43,420 But you don't know which mRNA, right? 723 00:44:43,420 --> 00:44:48,860 One hypothesis might be that you need the mRNA from M cyclin 724 00:44:48,860 --> 00:44:51,980 in order to produce cyclin every cell cycle. 725 00:44:51,980 --> 00:44:54,000 And that was their hypothesis. 726 00:44:54,000 --> 00:44:59,120 So what they did to test that was to degrade all the mRNA, 727 00:44:59,120 --> 00:45:02,660 inactivate the RNase, and then add back the mRNA 728 00:45:02,660 --> 00:45:05,627 to one gene, that mitotic cyclin. 729 00:45:05,627 --> 00:45:07,460 And what they saw when they did that is they 730 00:45:07,460 --> 00:45:10,310 restored the cell cycle, suggesting 731 00:45:10,310 --> 00:45:15,980 that this one mRNA, M cyclin, is sufficient to restore 732 00:45:15,980 --> 00:45:20,090 the oscillation of the mitotic cycle. 733 00:45:20,090 --> 00:45:23,070 OK, now, the last experiment, which 734 00:45:23,070 --> 00:45:25,920 I think is the most important, shows you the mechanism 735 00:45:25,920 --> 00:45:28,230 by which the cyclin is going. 736 00:45:28,230 --> 00:45:31,890 Because they added-- and instead of adding back 737 00:45:31,890 --> 00:45:34,590 the wild-type mitotic cyclin, they 738 00:45:34,590 --> 00:45:39,020 added back a cyclin mutant that was non-degraded all by this E3 739 00:45:39,020 --> 00:45:40,980 ubiquitin ligase mechanism. 740 00:45:40,980 --> 00:45:51,580 OK, so if they add back a cyclin mutant and this mutant has 741 00:45:51,580 --> 00:45:56,380 a deletion in the part of the protein called the destruction 742 00:45:56,380 --> 00:45:57,670 box-- 743 00:45:57,670 --> 00:46:00,310 and this is essentially the part of the protein 744 00:46:00,310 --> 00:46:06,430 that is recognized by the E3 ubiquitin ligase, OK? 745 00:46:06,430 --> 00:46:11,980 So the destruction box mutant basically blocks this such 746 00:46:11,980 --> 00:46:15,130 that cyclin is no longer polyubiquitinated 747 00:46:15,130 --> 00:46:18,890 and it can't be targeted for proteolysis. 748 00:46:18,890 --> 00:46:22,760 OK, and in this case, what happens 749 00:46:22,760 --> 00:46:25,340 is cyclin levels increase. 750 00:46:25,340 --> 00:46:29,600 And then they stay high and there's no cycle. 751 00:46:29,600 --> 00:46:33,770 OK, so when you have this cyclin mutant with the destruction 752 00:46:33,770 --> 00:46:36,980 box deleted such that it's non-degraded, 753 00:46:36,980 --> 00:46:39,845 it's not degraded, you get a cell cycle arrest. 754 00:46:43,870 --> 00:46:48,490 And because this is M cyclin, the cell arrests in mitosis. 755 00:46:48,490 --> 00:46:52,220 You get a mitotic arrest, a mitotic arrest. 756 00:46:56,110 --> 00:46:59,420 OK, any questions about this mechanism 757 00:46:59,420 --> 00:47:02,360 of proteolytic degradation? 758 00:47:02,360 --> 00:47:03,700 You all see how this-- 759 00:47:03,700 --> 00:47:04,820 yes, Malik? 760 00:47:04,820 --> 00:47:09,313 AUDIENCE: So [INAUDIBLE] cell [INAUDIBLE] what is it 761 00:47:09,313 --> 00:47:10,496 physically doing? 762 00:47:10,496 --> 00:47:12,450 ADAM MARTIN: What is it physically doing? 763 00:47:12,450 --> 00:47:15,390 It's basically stuck with a mitotic spindle 764 00:47:15,390 --> 00:47:18,340 and it's not segregating the chromosomes. 765 00:47:18,340 --> 00:47:21,000 Yeah, so it hasn't gone through mitosis. 766 00:47:21,000 --> 00:47:24,810 It's stuck in a specific phase of mitosis. 767 00:47:24,810 --> 00:47:26,460 In this case, it's stuck in basically 768 00:47:26,460 --> 00:47:29,100 a metaphase-like state. 769 00:47:29,100 --> 00:47:32,490 One last point I want to make about this 770 00:47:32,490 --> 00:47:36,760 is that the mRNA for M cyclin is just constant. 771 00:47:36,760 --> 00:47:38,380 It's always present. 772 00:47:38,380 --> 00:47:41,160 So this is constant. 773 00:47:41,160 --> 00:47:42,920 You have constant mRNA. 774 00:47:42,920 --> 00:47:44,670 CDK is constant. 775 00:47:44,670 --> 00:47:47,610 It's the cyclin protein that's going up and down. 776 00:47:47,610 --> 00:47:50,250 And it's going up and down because of this regulated 777 00:47:50,250 --> 00:47:53,100 proteolysis. 778 00:47:53,100 --> 00:47:54,690 OK, great. 779 00:47:54,690 --> 00:47:56,730 On Wednesday, we'll talk about stem cells 780 00:47:56,730 --> 00:47:58,970 and we'll talk about guts.