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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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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