Controlling the Cell Cycle
Transcript of Part 1: Controlling the Cell Cycle: Introduction
00:00:03.02 Hello, my name is Dave Morgan. I'm from the University of California in San Francisco. 00:00:07.07 And today we're going to talk about mechanisms of eukaryotic cell cycle control. 00:00:11.12 In this first introductory lecture I'm going to give you a basic overview 00:00:15.10 of the cell reproduction process and a little bit of information about 00:00:18.13 the regulatory system that guides the cell through that process. 00:00:22.09 And then in my second and third lectures, we'll go into some details about 00:00:25.23 experiments from my own laboratory. 00:00:29.09 So it's been known since the early 1800's 00:00:31.16 that all living things are composed of individual units called cells. 00:00:34.25 And so the growth, development and survival of all living things 00:00:38.19 depends on the reproduction of those cells. 00:00:40.22 So cell reproduction is clearly and fundamentally an important biological process. 00:00:46.17 Cell reproduction is also important for human disease. 00:00:49.07 One of the major diseases of the Western world, cancer, 00:00:51.23 is essentially a disease of excess cell reproduction. 00:00:55.12 This disease occurs when a cell in a tissue acquires a mutation 00:00:59.03 that allows it to proliferate more rapidly than its neighboring cells. 00:01:02.12 And through the acquisition of additional mutations, the progeny of that cell 00:01:06.08 can form into a tumor that can eventually escape the tissue, 00:01:09.06 as shown down here, and that results in malignant cancer spread throughout the body. 00:01:13.19 So an understanding of cancer absolutely requires an understanding of 00:01:17.26 the mechanisms of cell reproduction. 00:01:20.19 So the question is: How do cells reproduce? 00:01:23.01 And that's the questions we're going to address in my lecture today 00:01:25.01 and in my other two lectures. Where do new cells come from? 00:01:28.25 Well, this question has occupied scientists ever since those early days 00:01:32.11 when it was first realized that all tissues are made up of individual cells. 00:01:36.14 And in those early days, there were a number of competing theories 00:01:39.15 about how cells reproduce. In one hand, a number of prominent psychologists 00:01:44.13 of the early 1800's suggested that cells formed by so called "free cell formation", 00:01:48.26 whereby they essentially crystallized out of the intercellular fluid. 00:01:52.11 Another theory was that all cells are derived by the division of pre-existing cells. 00:01:58.08 And through 20 or 30 years of intense microscopy in a wide range of cells and tissues and organisms, 00:02:03.05 it became clear that this second theory was the correct one 00:02:05.27 and that, indeed, all cells are derived by the division of pre-existing cells. 00:02:10.08 In other words, cell reproduction is essentially a process 00:02:13.20 by which this remarkably complex machine, the cell, duplicates its contents 00:02:18.05 and then distributes those contents into a pair of genetically identical daughter cells. 00:02:24.00 Now this idea has profound implications because it means that all cells in existence today 00:02:29.12 are likely to be derived from a single, ancestral cell that divided perhaps 3.5 billion years ago. 00:02:36.29 So in the hundred years since the discovery that 00:02:39.04 all cells are derived from other cells by division, microscopists and, to a lesser extent, geneticists 00:02:44.09 dedicated their efforts to understanding the basic mechanics of the cell reproduction process. 00:02:49.07 And that led to our current view of what we call the cell division cycle, 00:02:53.03 which is illustrated in the next slide. So we now believe that 00:02:56.13 the cell cycle, or the series of events by which the cell duplicates itself, 00:02:59.22 is divided into a series of distinct phases that are typically 00:03:03.04 defined on the basis of chromosome duplication. 00:03:05.25 S phase is the period during which the chromosomes duplicate. 00:03:09.07 And M phase is the period during which those duplicated chromosomes, 00:03:12.20 or sister chromatids as they're called, are segregated to opposite poles of the cell 00:03:16.29 and then packaged into individual, genetically identical daughter cells. 00:03:20.14 Now, throughout this process, it's not just the chromosomes that duplicate, 00:03:24.19 but all the other components of the cell also duplicate. 00:03:27.12 All the proteins, organelles, RNAs and all the other macromolecular complexes of the cell 00:03:32.04 are all duplicated throughout...continuously throughout the entire cell cycle 00:03:36.07 so that by the time the cell divides at the end of M phase the resulting daughter cells 00:03:40.29 are essentially about the same size as the cell that first began that cell division cycle. 00:03:46.18 Now, M phase is a particularly spectacular phase of the cell cycle 00:03:49.19 that is typically defined as involving two distinct events-mitosis and cytokinesis. 00:03:55.21 Mitosis is the process by which the chromosomes 00:03:58.22 are segregated and packaged into individual nuclei. 00:04:01.12 And then cytokinesis is the process by which those daughter nuclei are then distributed 00:04:06.19 by cell division into a pair of genetically identical daughter cells. 00:04:10.14 The process of mitosis depends extensively on 00:04:13.07 this remarkable molecular machine called the mitotic spindle, 00:04:16.15 which is essentially a bipolar array of microtubule polymers 00:04:19.22 that forms as the cell enters mitosis 00:04:22.05 and then becomes attached to the sister chromatid pairs, 00:04:24.10 as I've shown here. The basic idea is that a mitotic spindle has these two poles 00:04:29.01 that radiate microtubules, some of which become attached 00:04:32.06 to the sister chromatid pairs, as I've shown here. 00:04:34.03 The attachment site on those chromosomes 00:04:36.28 is called the kinetochore, which is a large protein complex 00:04:39.23 that basically provides a microtubule binding site on the surface of the chromosome. 00:04:44.11 And so, by the middle of mitosis the chromosomes are aligned 00:04:49.14 along the middle of the mitotic spindle, as shown here, 00:04:52.01 with one sister chromatid attached to one spindle pole 00:04:54.23 and the other sister attached to the other spindle pole. 00:04:57.04 And so what happens next is that the glue, the protein glue, that holds 00:05:00.18 the sister chromatids together is then dissolved, 00:05:03.20 allowing those sister chromatids to be pulled apart by the spindle 00:05:06.15 in anaphase, here, and then packaged into individual nuclei 00:05:10.21 and then distributed by cytokinesis into the daughter cells. 00:05:17.15 This next slide gives us a little bit more detail about the process of mitosis, 00:05:21.00 the spectacular phase during which the chromosomes are segregated. 00:05:24.21 Mitosis typically begins with a stage called prophase which is shown in the upper left here. 00:05:29.10 Prophase is distinguished by the fact that chromosomes inside the cell, 00:05:34.05 inside the cell nucleus, begin to condense from their normally disperse state 00:05:37.23 into this much more compact rod-like structure that is more easily manipulated during mitosis. 00:05:43.10 In addition, prophase is also typified by changes 00:05:46.10 in the organization of the microtubule cytoskeleton 00:05:48.28 which is labeled here in green. 00:05:51.05 And so you can see that in prophase there are two microtubule organizing centers 00:05:54.22 and these two centers move apart from one another along the surface of the nucleus 00:05:58.20 to begin the formation of a mitotic spindle. 00:06:01.03 And then at the end of prophase, the nuclear envelope dissolves 00:06:05.10 allowing the microtubules of those organizing centers to gain access to 00:06:08.28 the sister chromatid pairs inside the nucleus until eventually the cell reaches metaphase 00:06:13.11 when those sister chromatid pairs are attached to the mitotic spindle, 00:06:16.11 as I said in the previous slide, in a bi-oriented fashion whereby 00:06:19.22 one sister is attached to one pole and the other is attached to the other pole. 00:06:24.13 And then the big event of mitosis occurs, which is when 00:06:27.01 the chromosome cohesion mechanisms 00:06:29.15 that hold those sister chromatids together are removed resulting in the separation of 00:06:33.18 the sister chromatid pairs and their movement to opposite poles of the mitotic spindle. 00:06:37.17 After which, in telophase, those separated chromosome sets 00:06:41.19 are then packaged into individual daughter nuclei. 00:06:44.11 After which, cytokinesis takes care of dividing the cell itself. 00:06:49.10 So the process of mitosis is really best appreciated by looking at movies of this process. 00:06:54.07 So this movie will show you the key event of mitosis 00:06:58.22 which is the metaphase to anaphase transition, 00:07:00.24 this point at which the chromosomes are pulled apart by the anaphase spindle. 00:07:05.08 And so this happens to be a vertebrate cell, 00:07:07.29 in which the microtubules are labeled in reddish-orange here 00:07:11.15 and then these green dots in the middle of the spindle 00:07:13.14 represent those kinetochore protein complexes 00:07:15.15 that join the sister chromatids to the microtubules in the middle of the spindle. 00:07:20.14 And so you can barely see these sister chromatids 00:07:23.06 as dark shadows in the middle of the spindle back here. 00:07:26.07 And so when I start this movie what we'll see is this metaphase cell 00:07:29.17 will move into anaphase through the separation of these sister chromatid pairs. 00:07:35.09 And so right about that instant right there, the sister chromatids are separated 00:07:40.20 and then pulled apart by the anaphase spindle. And we'll see it one more time. 00:07:48.17 OK, so that is mitosis in the context of a cultured cell on a plastic dish. 00:07:53.08 What this movie will show you is mitosis in the context of a living organism, 00:07:58.08 in this case, the early embryo of the zebra fish. 00:08:00.14 And in this case the zebra fish embryo has been filmed using a modern microscopy technique 00:08:05.18 that allows the identification of all the individual nuclei in that embryo. 00:08:09.18 And so in this image right here, for example, this first image, 00:08:12.17 we're looking at a zebra fish embryo early in development where it has 00:08:15.22 about 20 or 30 nuclei in it, which have obviously undergone a number of divisions. 00:08:19.25 And each of those nuclei is visible as one of these little, white blobs 00:08:23.10 that essentially represents the fluorescently labeled DNA in the nucleus. 00:08:26.23 And so when I start this movie you'll see how these little DNA blobs, or nuclei 00:08:32.07 rapidly divide and subdivide again and again to populate this embryo with the large numbers of cells 00:08:37.27 that are needed for embryogenesis. 00:08:41.12 And so, we can see this incredible division process occurring over and over and over again 00:08:45.21 in this embryo. Resulting in the transformation of those 20 or 30 cells 00:08:49.11 into the thousands of cells that are required to populate this embryo. 00:08:52.29 And as time goes by, over the next few minutes 00:08:56.11 we begin to see the migration of these cells to form the basic pattern of the early embryo. 00:09:03.15 So clearly, this movie emphasizes the key point that cell division and reproduction 00:09:08.08 is a crucial process in the development of all living things. 00:09:13.05 OK, so that gives you a basic overview of the basic mechanics of cell division 00:09:17.29 and what I want to do for the rest of this lecture and for my other lectures as well 00:09:21.09 is focus on a more recent problem that has occupied scientists for the last 20 or 30 years or so. 00:09:26.01 And that is the question or the problem of how these remarkable cell cycle events are controlled 00:09:30.15 such that they occur always in the correct order 00:09:33.16 and with the appropriate timing and coordination. 00:09:35.27 Why, for example, S phase always occurs before M phase and so on. 00:09:39.15 And the answer to this question has turned out to be that the cell contains this remarkably 00:09:43.12 complex regulatory system that guides the cell through the stages of cell division. 00:09:47.28 And this complex regulatory system has occupied scientists, as I said, 00:09:52.24 for about 20 or 30 years at this point. 00:09:55.21 Studies of this regulatory system have come from a wide range of different model organisms 00:10:00.02 and, in fact, most of the major discoveries about this system 00:10:02.21 have come from very simple eukaryotic organisms such as the budding yeast 00:10:06.27 and fission yeast, as I'll show you in the next slide. 00:10:10.07 So this slide illustrates a few of the really important model organisms systems 00:10:14.16 for studies of cell cycle control. In the upper left, is the budding yeast Saccharomyces cerevisiae 00:10:20.01 and below that the fission yeast Schizosaccharomyces pombe. 00:10:23.13 And these two yeasts have turned out to be extremely important 00:10:26.07 model systems for studying the basic features of cell division 00:10:28.29 for many reasons. First of all, their regulatory mechanisms for cell cycle control 00:10:33.19 are very similar to the same mechanisms that are found in human cells. 00:10:37.18 They also have a number of extremely important experimental advantages. 00:10:41.19 They have short generation times, completely sequenced genomes for quite some time. 00:10:45.18 It's possible to do a wide range of genetic manipulations in these organisms 00:10:50.02 and most importantly, it's possible to apply the methods of classical genetics 00:10:53.13 to these organisms to isolate mutant genes that allow 00:10:57.00 the analysis of the principles and the components involved in cell cycle control. 00:11:01.21 And so the budding yeast and the fission yeast have been really important 00:11:05.23 tools in our study of cell cycle control. 00:11:09.05 Another important organism is shown along the bottom here, 00:11:11.08 and this is the early embryonic divisions of the frog. 00:11:14.20 Early embryonic cells both in frogs, and in vertebrates as well, 00:11:18.27 have been extremely important in studies of cell cycle control 00:11:22.11 because these early embryonic divisions represent simplified, 00:11:26.05 stripped down versions of the cell cycle, essentially, 00:11:28.06 that can be analyzed very easily biochemically for a variety of reasons. 00:11:33.03 And then finally, some other organisms that have proven to be important are 00:11:36.13 the early embryo of the fruit fly Drosophila melanogaster, which has been particularly 00:11:41.14 crucial for studies of cell cycle control in early embryonic development. 00:11:44.24 And then here of course, is a mammalian cell growing in culture 00:11:48.15 which is also a very important tool in which to translate some of 00:11:52.11 the findings that have been found in some of these other organisms into the context of 00:11:55.20 a human cell. This next slide goes into a little bit more detail about the budding yeast 00:12:02.04 and its cell cycle because this particular organism has been 00:12:05.15 so instrumental in cell cycle control studies and also because 00:12:07.22 it is the organism we study in my lab and you'll need to know some 00:12:10.15 of this for my second and third lectures. 00:12:13.22 So budding yeast, as the name implies, divides by budding. 00:12:16.02 Which means that at the end of G1, when this cell enters the cell cycle, 00:12:20.02 it starts growing a little bud on the side of the mother cell 00:12:23.01 and as the cell progresses through the cell cycle, the size of that bud increases 00:12:27.13 until by the end of mitosis the bud is almost the same size as the mother cell. 00:12:32.04 And so the size of the bud in budding yeast gives a very good indication 00:12:35.23 of the position of that cell in the cell cycle. 00:12:37.29 And this turned out to be a very useful tool because it then allowed the isolation of 00:12:42.20 mutants that were arrested at specific cell cycle stages. 00:12:45.13 And so Lee Hartwell and his colleagues back in the late 1960s and early 1970s 00:12:50.12 isolated a wide range of mutants in various genes that resulted in 00:12:55.09 cell cycle arrest at specific stages that were determined on the basis of bud morphology. 00:13:01.07 These mutants were conditional temperature sensitive mutants 00:13:03.23 which means that at normal room temperature these mutant genes were 00:13:07.14 totally functional and the cell divides, 00:13:09.07 but at high temperature, 37 degrees, these mutant genes, 00:13:12.25 or rather the gene products, become inactivated and as a result 00:13:16.01 the cell then arrests at the cell cycle stage where that gene product is required. 00:13:20.08 And a couple examples of those sort of mutants are shown in the next slide. 00:13:24.06 These were the so called cell division cycle or cdc mutants and two of them are shown here. 00:13:30.01 On the left is cdc16, a mutant that causes at the high temperature an arrest in mitosis. 00:13:36.13 As you can see by the size of this bud, this cell is arrested in mitosis. 00:13:39.16 However, you can see from DNA staining here, that the cell 00:13:43.17 has not yet segregated its chromosomes. 00:13:45.10 So this cell is arrested before anaphase. 00:13:47.29 And then looking at the spindle gives us another clue that 00:13:50.17 this cell actually contains a short metaphase spindle. 00:13:53.20 And so together, these various phenotypes indicate that cdc16 mutants 00:13:57.11 arrest in metaphase prior to the onset of anaphase. 00:14:00.24 And indeed, it turned out since the discovery of this mutation 00:14:03.18 that cdc16 encodes a component of an important protein complex 00:14:08.05 that is required for progression through the metaphase to anaphase transition. 00:14:13.08 And so cdc15, in contrast, is a little different. It's also a mitotic arrest 00:14:18.00 but in this case the DNA has segregated into two distinct masses. 00:14:21.11 And so...and by looking at the spindle in these cells it's clear that these cells 00:14:25.23 have reached anaphase but have not progressed any farther than that. 00:14:28.29 They contain the long anaphase spindle, segregated DNA, but 00:14:33.06 they've arrested at the end of anaphase. And this turns out to be because 00:14:37.21 cdc15 encodes a component of a regulatory network that drives the cell 00:14:42.04 out of anaphase and into the following G1. 00:14:45.14 And so by isolating and characterizing large numbers of these cdc mutants, 00:14:49.04 yeast geneticists working in budding yeast and in fission yeast, 00:14:53.01 with the work of Paul Nurse and colleagues, 00:14:54.23 led to the isolation of a large number of these cdc mutants that 00:14:58.12 have essentially built the foundation for our present understanding of cell cycle control. 00:15:06.05 The other major organism that we need to discuss in a little detail is the frog, Xenopus laevis, 00:15:11.16 whose early embryos have turned out to be an extremely important 00:15:13.25 tool for studies of cell cycle control as well. 00:15:16.18 Following fertilization, the egg...the fertilized egg of the frog 00:15:20.13 divides rapidly in these so called cleavage divisions which 00:15:23.29 transform that large fertilized egg into thousands of cells in a matter of a few hours essentially. 00:15:30.12 And it goes through these very simplified versions of the cell cycle 00:15:33.18 that have only S and M phases and no gap phases. 00:15:36.15 And these occur over a period of about 30 minutes each. 00:15:39.14 And so this very rapid, simple cell cycle has turned out to be an important 00:15:43.15 tool for these studies of the basic features of cell cycle control. 00:15:47.18 One big advantage of frogs and their embryos is that these eggs are so large 00:15:51.23 that they can be injected very easily with test substances 00:15:54.15 or for that matter it's possible to isolate large amounts of the cytoplasm 00:15:57.29 from these fertilized eggs and recreate the events of the cell cycle in a test tube. 00:16:05.08 And so it was these early studies of yeast genetics on one hand and frog embryonic cells on the other 00:16:10.04 that led to a couple of competing or alternative views of how cell cycle control is achieved. 00:16:14.26 And those are illustrated in this slide. 00:16:18.02 On the left is the view that resulted from studies in yeast and 00:16:20.25 particularly from various studies of cdc mutants like the ones I showed you. 00:16:24.12 Those studies suggested that many cdc mutants arrest...when they arrested 00:16:28.21 the cells in early cell cycle stages also blocked progression through later cell cycle stages. 00:16:33.15 And so, for example, a cdc mutant that arrests cells prior to DNA replication 00:16:37.23 also caused a block to the onset of mitosis. 00:16:42.04 And so this suggested that there were these dependency relationships 00:16:44.09 among the events of the cell division cycle such that DNA replication, for example, 00:16:49.02 has to occur before mitosis can occur. 00:16:52.14 And so the idea was that one event, such as event A or DNA replication, 00:16:56.06 must be completed in order for event B to begin. 00:16:59.23 And so these events might some how regulate each other in some way. 00:17:04.06 Now, the alternative view, on the right here, 00:17:06.23 came from studies in the early embryos of the frog which 00:17:09.23 clearly suggested that these frog eggs, these early frog embryos, I should say 00:17:14.04 contain an intrinsic biochemical timer that turns on cell cycle events 00:17:19.08 at specific times and in a specific order. 00:17:21.16 In other words, the events of the cell cycle and their order and timing 00:17:25.10 are determined by a programmed timer or a clock that essentially flips switches 00:17:29.28 at specific times to initiate the cell cycle events. 00:17:32.25 And so that timer is independent of the events that it controls. 00:17:36.11 And in fact, in frog embryos it's possible to block DNA replication or 00:17:40.00 completely remove the nucleus and still see evidence 00:17:42.05 that this timer is operating normally and oscillating. 00:17:46.19 So how do we reconcile these two different models? 00:17:48.25 Well, the answer turned out to be that both were at least partially correct. 00:17:51.28 There is, indeed, an intrinsic biochemical timer in all eukaryotic cells 00:17:55.20 that guides the cell through the cell division cycle, but in many cells 00:17:58.27 the events of the cell cycle can feed back 00:18:01.08 to that timer and adjust the timing of later events 00:18:03.28 if early events fail for one reason or another. 00:18:06.13 And that's summarized in this next slide. 00:18:10.22 So, once again, the basic idea is that the eukaryotic cell cycle is 00:18:14.20 governed by this intrinsic biochemical timer or control system 00:18:17.29 that essentially, is a series of biochemical switches that turn on specific cell cycle events 00:18:23.19 in a specific order and in a specific time. 00:18:26.28 However, that timer can be regulated in such a way that those cell cycle events 00:18:30.23 can feed back and send information to the timer to arrest progression through certain stages 00:18:35.05 if necessary. And so there are three major transitions or so called checkpoints in the cell cycle 00:18:40.21 where this timer can be arrested if conditions are not appropriate. 00:18:45.29 And so for example, if the timer initiates S phase as shown here, 00:18:49.09 but for some reason DNA synthesis fails and a chromosome fails to duplicate, 00:18:53.03 then that will send a message back to the controller that will block progression through 00:18:57.11 the G2/M checkpoint over here. So the timer will basically arrest 00:19:00.08 at this point here, thereby preventing 00:19:02.28 entry into mitosis if the chromosomes are not fully duplicated. 00:19:06.12 And likewise there are similar mechanisms 00:19:09.07 in operation at the metaphase-anaphase transition 00:19:11.08 and at the start or G1/S checkpoint at the beginning of the cell cycle. 00:19:14.23 At all of these checkpoints or transition points it's possible to arrest 00:19:18.00 the timer under certain conditions if certain previous events are not carried out successfully. 00:19:24.29 OK, so with this basic idea of cell cycle control in mind 00:19:28.18 the big question that has occupied many of us for the last 10 or 20 years 00:19:31.22 has been understanding what the molecular composition of this timer is. 00:19:35.17 What are the proteins that make up this timer and 00:19:38.19 are assembled together into this remarkable regulatory system? 00:19:42.04 And here again, the major breakthroughs in this field 00:19:44.28 arose out of a combination of both yeast genetics and 00:19:47.22 biochemical studies primarily in early embryonic cell types. 00:19:52.02 And that all collided essentially, in the late 1980's when it was realized that 00:19:56.03 people working in yeast genetics and people working in early embryos 00:19:59.25 of frogs and other systems 00:20:01.04 were actually studying essentially the same regulatory molecules. 00:20:05.06 In budding yeast for example, a protein kinase called Cdc28 was found to be required for 00:20:11.12 entry into the cell cycle. In fission yeast, a highly related protein kinase called Cdc2 00:20:16.25 was identified that was required for progression into mitosis. 00:20:20.21 And these two protein kinases, Cdc2 and Cdc28 appeared to be the homologues of one another 00:20:25.20 in these two species and were clearly crucial regulators of cell cycle progression in these yeasts. 00:20:31.11 Meanwhile, in the frog, Masui and others had identified a Maturation Promoting Factor 00:20:36.09 or MPF activity which appeared to be capable of driving mitotic eggs 00:20:42.01 of the frog into mitosis. And so this so called M-phase promoting factor was capable of 00:20:47.04 initiating mitosis when injected into non-mitotic cells. So that clearly represented 00:20:53.25 some sort of important biochemical activity that might be important for cell cycle control. 00:20:57.20 And then finally, Tim Hunt and colleagues identified a protein 00:21:01.01 called Cyclin whose levels oscillate during 00:21:03.09 the cell cycle and rise in mitosis and fall there after suggesting that 00:21:06.24 it might be some component of an important regulatory molecule. 00:21:10.28 And so as I said, in the late 1980's all these fields collided when it was realized 00:21:15.01 that all of these people were actually working on the same thing 00:21:19.10 and that MPF for example, this Maturation or M-phase Promoting Factor was 00:21:22.26 in fact, a complex of a small protein kinase that was related to Cdc28 and Cdc2 and Cyclin. 00:21:28.28 And so a complex of Cdc28 or Cdc2 and a Cyclin molecule forms a protein kinase 00:21:34.21 complex that is responsible for driving progression through the major stages of the cell cycle. 00:21:40.00 And that's illustrated in the next slide. 00:21:41.23 The basic idea is that the heart of the control system that guides 00:21:45.25 the cell through cell division is a protein kinase called a Cyclin-dependent kinase or Cdk. 00:21:51.23 And Cdks are inactive until they associate with their Cyclin regulatory partners, 00:21:55.28 resulting in the activation of a Cdk-cyclin complex. 00:22:00.11 Those Cdk-cyclin complexes then phosphorylate large numbers of substrates in the cell 00:22:05.08 resulting in the onset of various specific cell cycle events. 00:22:11.22 And so our current view of this system is that there aren't simply 00:22:16.05 one or two Cdks in the cell but in fact there are series of Cyclin-Cdk complexes 00:22:20.26 each of which is responsible for driving the specific events of the cell division cycle. 00:22:25.04 And so for example, a complex of Cdk with an S-phase Cyclin forms in late G1 and 00:22:30.09 is activated at that point to trigger the onset of DNA replication. 00:22:34.27 And then likewise, later in the cell cycle, an M-phase cyclin-Cdk complex forms and 00:22:38.29 is activated, resulting in the initiation of mitotic entry. 00:22:43.07 And so through this series of Cdk-cyclin complexes, the events of the cell cycle are 00:22:47.27 triggered in the appropriate order and with the appropriate timing. 00:22:52.13 Of course, since the initial discovery of Cdks it's become clear that they're 00:22:56.14 activities are controlled by far more that just the Cyclin regulatory subunit. 00:23:01.03 Cdks, both the mitotic and S-phase sort are also regulated by phosphorylation 00:23:06.26 on the Cdk subunit and also by inhibitory proteins that can associate with Cdks 00:23:10.23 at certain cell cycle stages to restrain their activity. 00:23:13.28 And so, Cdk activity and the up-regulation and down-regulation of Cdk activity 00:23:20.03 during the cell cycle depends on a wide range of different regulatory mechanisms. 00:23:26.21 So, the next question following the discovery of Cdks 00:23:29.23 dealt with this interesting observation over here 00:23:33.12 which is that Cyclin levels drop precipitously during mitosis. 00:23:37.14 And in fact studies, a wide range of studies, suggested that the destruction of 00:23:41.08 Cyclins in mitosis is actually required for progression out of mitosis. 00:23:45.19 So for example, if you make a version of Cyclin that cannot be degraded, that is highly 00:23:48.25 stabilized and remains high throughout mitosis, cells expressing that stabilized Cyclin 00:23:54.21 fail to exit. They fail to get out of anaphase and exit into the following G1, 00:23:59.10 indicating that Cyclin destruction is in fact required for the exit from mitosis. 00:24:04.11 And so a lot of effort was placed on identifying the mechanisms 00:24:07.14 that determined this Cyclin destruction. 00:24:09.04 And that led, primarily through biochemistry with a little bit of yeast genetics on the side, 00:24:12.26 led to the identification of a large protein complex that is responsible 00:24:17.00 for the destruction of cyclins. And that protein complex is called 00:24:20.28 the Anaphase-Promoting Complex or Cyclosome, otherwise known as the APC. 00:24:25.04 And its activity rises in mid-mitosis and is responsible for destroying cyclins. 00:24:31.22 The APC is not a protein kinase but a ubiquitin-protein ligase or E3 which means that its job 00:24:37.18 is to catalyze the attachment of a small protein called ubiquitin onto its targets. 00:24:42.04 And by attaching large numbers of these ubiquitins onto its targets 00:24:45.18 that sends those targets to a protease in the cell called the proteasome 00:24:49.02 where they are destroyed. And so the APC is essentially capable of triggering 00:24:52.26 the destruction of its target proteins, including the cyclins. 00:24:57.15 The next slide illustrates in a little bit more detail what it is that the APC does. 00:25:01.10 Now, as I mentioned, the major target of the APC is the Cyclin of this Cdk regulatory subunit. 00:25:08.26 And so the destruction of cyclins results in the inactivation of Cdks in late mitosis. 00:25:13.02 The other major target of the APC is a protein called securin. 00:25:17.06 And securin is a tight binding inhibitor of a protease called separase. 00:25:22.14 And so when securin is destroyed in mitosis by the APC that results in 00:25:27.02 the liberation of separase in the cell and separase then goes to the sister chromatids 00:25:31.10 and cleaves a single subunit of a protein complex called cohesin 00:25:35.04 that holds those sister chromatids together. 00:25:38.01 And by cleaving that subunit, separase induces the separation of the sister chromatids 00:25:42.04 which can then be segregated by the microtubules of the mitotic spindle. 00:25:46.08 And so the APC, through this mechanism, directly triggers the initiation of anaphase. 00:25:52.11 Now, as I said, it also triggers the destruction of cyclin and therefore 00:25:55.17 the inactivation of the associated Cdks, and this turns out to be very important, as I mentioned 00:26:00.06 because this allows the dephosphorylation of substrates of the Cdks. 00:26:05.12 And this dephosphorylation of Cdk substrates is required for 00:26:08.22 normal progression out of mitosis. 00:26:10.27 Normal anaphase and normal telophase and cytokinesis and mitotic exit all depend on 00:26:16.12 the dephosphorylation of Cdk substrates. 00:26:19.08 And so, cyclin destruction is therefore crucial, one of the crucial jobs of the APC in late mitosis. 00:26:26.28 And so we're left with this general scheme of cell cycle control 00:26:30.08 whereby the cell cycle control system is essentially a series of biochemical switches 00:26:35.05 made up of cyclin-dependent kinases of various sorts 00:26:38.00 that turn on the various events of the cell cycle until the cell reaches metaphase 00:26:42.05 when the chromosomes are aligned on the mitotic spindle. At which point the APC 00:26:46.05 triggers the destruction of securin and cyclins to take the cell out of mitosis 00:26:50.09 and complete the cell cycle. 00:26:52.20 Now obviously, as I said before, this is a highly simplified view of things 00:26:55.16 because there are countless other regulators that are sending 00:26:58.23 inputs into this system to refine the activation of Cdks and the APC in various ways. 00:27:03.27 For example, there are numerous phosphorylation events on the Cdks and on the APC itself 00:27:09.08 as well as various inhibitory proteins that bind to those proteins. 00:27:13.00 Now, recent studies have begun to focus... 00:27:15.02 now that we have the basic components of the cell cycle control system in place, 00:27:18.06 have begun to focus on how these components are assembled into a regulatory system that 00:27:22.17 achieves the behaviors that we're so interested in. 00:27:26.05 So this field is now reaching into the systems biology field 00:27:29.01 and beginning to use mathematical modeling to put these components into a regulatory system 00:27:34.03 and model that system to see how it achieves the various behaviors that it achieves. 00:27:38.09 So for example, one key question is 00:27:40.23 the kinetics of Cdk activation at the beginning of S-phase and M-phase. 00:27:45.10 It's well established that these kinases are both activated in a highly 00:27:49.06 switch-like fashion at the onset of S-phase or the onset of M-phase. 00:27:52.22 And so, in other words, their activity goes from very low activity to very high activity very rapidly. 00:27:59.21 And so there are biochemical mechanisms such as positive feedback loops involved 00:28:02.28 that ensure that these Cdks go from low to maximal activity abruptly. 00:28:07.15 And this, of course, allows the complete commitment of the cell 00:28:10.22 to a new cell cycle stage through this abrupt activation of Cdks. 00:28:15.19 And so, that gives you an overview of the basic features of the cell cycle control system 00:28:20.21 and some of the issues that are attracting scientists today 00:28:23.09 and it prepares you for my next two lectures 00:28:25.22 in which I will address some of these issues in a little bit more detail.