Eukaryotic Cell Division
Transcript of Part 1: Separating Duplicated Chromosomes
00:00:09.22 Hello. My name is Dick McIntosh. I am a 00:00:11.15 cell biologist from the University of Colorado. 00:00:14.08 I study cell division, and I've been working on 00:00:16.29 that problem for about forty years. 00:00:19.08 It's a fascinating problem because cells 00:00:23.22 must divide in order to live. 00:00:25.24 Cells are very complicated, so the division process is itself complex 00:00:30.11 because every time a cell divides you basically 00:00:32.23 have to build two new cells where you previously just had one. 00:00:37.13 This means of course there's a lot of biosynthesis that must go on, 00:00:40.21 in order to provide the materials that will allow 00:00:44.17 a cell to produce two viable daughters. 00:00:46.26 Now cells are very complex. 00:00:49.27 Their complexity can be thought of in terms of the number of instructions 00:00:55.06 that it takes in order to build a cell. 00:00:57.07 And although we don't know that number precisely, 00:00:59.26 you can estimate it from the number of genes 00:01:03.13 and from the ways in which genes are regulated, 00:01:06.00 and from existing proteins in the cell. 00:01:08.10 And in short, the number of instructions that's involved 00:01:11.10 to build a cell is more than the number of instructions that it takes to build 00:01:15.04 even a very complex manmade object, 00:01:18.07 such as a moon rocket or supercomputer. 00:01:20.17 This means that cells really are the very high end of complexity 00:01:25.04 in terms of the kinds of structures and systems 00:01:28.12 that we think about. 00:01:29.22 But on the other hand, cells are very small. 00:01:32.15 They're so small that if you take cells from say, the human liver, 00:01:37.07 it takes something like a million of them 00:01:38.29 to make an object as big as a pinhead. 00:01:41.07 And this means that they are not only highly specialized 00:01:44.20 in terms of their complexity, but also in terms of 00:01:48.21 their micro miniaturization. Now on top of this, 00:01:52.05 cells can reproduce themselves. 00:01:54.16 Imagine what you would have if you had a self reproducing 00:01:57.06 moon rocket or super computer. 00:01:58.24 But cells can achieve this remarkable goal, and this is the process 00:02:03.09 that we are going to look at in three lectures. 00:02:05.25 In this first one, I am going to give background information. 00:02:09.12 To some extent, it is textbook information, 00:02:12.22 but I hope I can put an interesting light on it 00:02:14.08 for those of you who already know quite a bit about the subject. 00:02:17.14 In the second lecture, I am going to describe 00:02:19.10 the experiments that have been done 00:02:21.15 by a wide variety of laboratories 00:02:23.17 including my own, in order to try to probe the machinery 00:02:27.15 that allows the cell to divide. 00:02:29.16 And in the final lecture, I'll talk about some recent work from our lab 00:02:32.29 in which we are trying to understand the actual mechanism by which 00:02:37.02 chromosomes move to the poles when a cell is dividing. 00:02:40.18 So, let's start thinking about this in terms of 00:02:45.22 the structures that you have in a cell that have to be reproduced. 00:02:50.10 If you look at this electron micrograph, 00:02:53.04 you see of course the familiar nucleus, 00:02:55.00 the cytoplasm, with all its complex membranes. 00:02:57.21 Not shown here, are all the complexities of the cytoskeleton as well. 00:03:02.02 All of these structures that we can see in a cell, 00:03:05.15 are actually made from the assembly of macromolecules, 00:03:09.13 proteins in most cases, sometimes nucleic acids, 00:03:11.14 carbohydrates, and lipids. And all of these constituents have to be 00:03:16.02 synthesized before the cell is ready to go into division. 00:03:19.12 So preparation for division involves a tremendous amount of biosynthesis. 00:03:25.07 The instructions for this division come by and large from 00:03:29.09 the DNA, which is located in the nucleus, 00:03:32.10 and has the familiar base pair structure that 00:03:35.27 allows the sequence of the nucleic acids 00:03:39.10 to define the sequence of a message, which in turn 00:03:42.03 defines the translated product. 00:03:44.01 And yet, that complexity of itself is not sufficient for building a cell. 00:03:49.24 because you could take DNA and put it in a test tube and transcribe it 00:03:53.19 and translate it, and you still won't 00:03:55.14 build a cell. It requires a cell to build a cell, 00:03:58.20 and so what we find in the structure of a cell 00:04:02.09 is a template in effect, which allows 00:04:04.14 those individual gene products as they are made to 00:04:06.13 go ahead and assemble in place in order to build the structures 00:04:10.19 that are necessary. Now you can watch this process 00:04:13.19 in phase microscopy looking at something like these two cells 00:04:17.28 shown here imaged in my lab a long time ago. 00:04:20.19 It's a time lapse movie, which compresses about twenty hours 00:04:26.11 of a cell cycle into about thirty seconds. 00:04:28.11 So time is flying in here, and you can watch 00:04:31.15 any one individual cell, and as you watch it moving around, you'll see 00:04:35.22 it divide. The periods between division, which are of course, called 00:04:39.20 interphase, are the times of synthesis that I've been talking about. 00:04:42.20 It's then that proteins are made, RNA is made, 00:04:47.04 DNA is replicated, so that we have two cells in one bag. 00:04:51.14 The process of cell division then is the mechanics of separating 00:04:56.07 all those constituents into two discrete objects. 00:04:59.08 And it has to be done very well, because 00:05:01.25 if cells lack essential constituents, for example 00:05:05.16 a chromosome, some of the DNA which includes instructions 00:05:09.04 for making RNA and protein. 00:05:11.24 Then the daughter cells will be unlikely to survive. 00:05:14.17 So the division process is a very precise one, 00:05:18.28 that has to take advantage of structures that are built 00:05:23.00 in order to ensure the accurate segregation of 00:05:25.29 the components of the cell, which are present in only a few numbers, like chromosomes. 00:05:30.13 What you are seeing here is cell division going forward with no restraint. 00:05:37.05 The cells are being provided with the factors 00:05:39.12 to stimulate their division. They are being provided 00:05:41.24 with all the food that they need. 00:05:43.10 and so they simply divide as rapidly as they can complete 00:05:46.20 the essential synthetic processes. 00:05:48.01 This is like the life style of a unicellular organism 00:05:52.23 where food is really the limiting factor 00:05:56.24 in how a cell can go forward making 00:05:59.12 decisions to divide and produce two of itself. 00:06:02.14 For the cells in our body on the other hand, this process 00:06:05.28 must be tightly regulated 00:06:07.23 because if cells divide too frequently you can get anomalies 00:06:12.16 which are very dangerous medically, like cancer. 00:06:14.27 On the other hand cells must divide 00:06:17.01 in order to achieve a healthy adult organism. 00:06:20.14 Of course, the cells of an embryo divide in order to produce 00:06:24.09 the juvenile and then the adult form. 00:06:25.29 Cell division is essential in wound healing. Cell division is also essential in tissue renewal, 00:06:33.10 so for example the red blood cells that circulate through 00:06:37.03 your vasculature are comparatively short in their lifetime, 00:06:42.00 a couple of weeks, and if they are not replaced, 00:06:44.13 you will have anemia. So cell division is being 00:06:48.03 regulated in an multicellular organism 00:06:49.27 to produce all the cells that are necessary to balance 00:06:54.19 the cell death which is occurring. 00:06:56.25 This balance of cell division and cell death 00:06:59.16 is a big subject in its own right. That is not what I am going to be talking about now. 00:07:04.06 What I am going to be talking about is the ways in which cells 00:07:07.06 go ahead and synthesize during these various periods of interphase 00:07:12.29 the constituents they need in order 00:07:14.27 to be able to achieve an accurate cell division. 00:07:19.12 The synthesis of course includes DNA synthesis, 00:07:21.13 in the S phase of the cell cycle, but there's a 00:07:24.11 gap before that and a gap afterwards and both of these are times of continued protein 00:07:29.21 synthesis, RNA synthesis, and cell growth, 00:07:33.00 so that when the cell finally comes to make a decision 00:07:36.05 to enter the division process, it is already fully 00:07:40.13 two cells in one bag, and the division process itself is simply a mechanical 00:07:46.03 division of all those constituents. 00:07:48.16 Now when a cell is going to try to divide, 00:07:52.14 it faces a series of problems. 00:07:54.12 As I have been emphasizing, there must be enough of all of its components 00:07:58.19 that it can go ahead and provide for the daughter cells. 00:08:01.07 There must be enough ribosomes and mitochondria, 00:08:04.25 and individual enzymes, and so forth. 00:08:07.12 These are objects that are present in large numbers. 00:08:10.28 And the strategy that a cell has for handling these 00:08:13.21 at a division is that if there are enough copies of 00:08:16.27 and individual object, like a ribosome, 00:08:18.18 the chances that one daughter cell will get all of them 00:08:22.14 are very small and so simply pinching the cell in the middle 00:08:26.07 will be sufficient to ensure that each daughter gets plenty. 00:08:30.01 But there are other structures in the cell 00:08:32.06 which are present in small numbers of copies, 00:08:35.14 chromosomes are an example, the centrosomes are an example, 00:08:38.24 also, the organizers for the microtubule component of the cytoskeleton. 00:08:42.22 Here you have one or two copies of each object, 00:08:46.22 they duplicate during interphase, and in order to ensure that the daughter cells 00:08:51.13 will get everything it needs to divide, 00:08:53.19 we have to have a special machine that is going to ensure 00:08:57.21 equal partition in the time preceding cell division. 00:09:01.20 Now that process of equipartitioning and cell division 00:09:06.15 is the process of forming the so called mitotic spindle to segregate the chromosomes. 00:09:12.24 And this is a structure that is familiar to all of us from the spring of our scientific 00:09:18.03 career, and yet it is a wonderfully complex structure, 00:09:21.25 that is remarkably accurate in its ability to do its job. 00:09:25.02 It forms in the cytoplasm off of organelles, the centrosomes in animal cells 00:09:31.15 and less well defined structures in some other cell types. 00:09:34.11 And it represents then a family of microtubules that will grow into 00:09:40.21 the region of the nucleus where the chromosomes have been condensing. 00:09:44.20 It will then attach to the chromosomes and organize them 00:09:48.12 and the organization puts them into the structure of the metaphase plate. 00:09:54.06 Chromatids then will segregate and we will get then this anaphase process 00:09:59.18 which involves chromosomes moving to the poles 00:10:01.27 and the elongation of the spindle so that later on the cell can simply pinch in the middle 00:10:08.03 and ultimately give rise to two cells. 00:10:10.24 So the mitotic spindle is the structure which is this special machine. 00:10:15.15 It segregates the chromosomes and it segregates the centrosomes. 00:10:18.29 And that's what we are going to try to understand. 00:10:21.28 The job that the spindle faces is an extremely difficult one 00:10:26.15 because eukaryotic cells in particular 00:10:28.21 have a huge amount of DNA. There are many, many bases, 00:10:34.01 all arranged in long strings and these 00:10:36.26 strings are in fact long enough that they make polymers that are millimeters to 00:10:41.11 even meters in length, whereas the cell is measured in many micrometers in length. 00:10:47.00 So there is a factor of thousands in the length scale difference 00:10:52.03 between the polymers that we need to segregate and 00:10:55.02 the cell itself, which means that the DNA must be all bundled up in some way 00:10:59.09 within the cell. And these chromosomes, one or more, 00:11:03.07 have to be segregated accurately if we are going to get all the DNA 00:11:06.29 into the daughter cells, and this must occur for the viability of the daughters, 00:11:12.23 because the loss of even a single chromosome is generally lethal. 00:11:16.11 You lose so many genes that the cell just cannot survive. 00:11:20.01 This set of micrographs brings us back to the nucleus with which we started. 00:11:25.10 It shows that if you allow the DNA to spill out, and you wash it clean of some of its proteins, 00:11:31.22 you can see what a tremendous extent of material it is. 00:11:35.13 This long extent is what we need to duplicate, 00:11:40.06 and our problem is that it is about five thousand times longer than the diameter of the cell 00:11:45.04 and how do we achieve this process? 00:11:47.28 The solutions that cells have come up with in order to solve this problem are 00:11:52.28 numerous. One of them is that the DNA is packaged in more than one piece, as a rule 00:11:57.14 in eukaryotes anyways, and this means that we have divided up all that DNA into 00:12:02.18 smaller segments. The DNA's always replicated before cell division 00:12:08.03 begins. This means that in eukaryotes we have a situation 00:12:12.06 where the whole set of double DNA 00:12:16.07 is ready for us to operate on. 00:12:18.12 Another trick that the cell uses is to keep 00:12:22.12 these sister chromatids as they are called 00:12:25.10 the duplicated DNA double helices fastened together so that the two identical 00:12:31.07 pieces of DNA are linked non-covalently by a protein complex, 00:12:37.26 and it is going to keep them in order while the cell is getting ready to divide. 00:12:42.11 The other point is that the chromosomes will condense tremendously, decreasing 00:12:48.17 their length down to make them an object that is small enough 00:12:51.25 that its full extent is less than the diameter of the nucleus. 00:12:55.25 So there's a tremendous amount of compaction 00:12:58.23 and finally we will develop that special machine, the mitotic spindle, 00:13:02.19 which could do the segregation job. 00:13:04.15 Now when DNA is replicating, what you have 00:13:08.11 is here a piece of DNA which is a circle, this is actually 00:13:12.25 a viral genome, and here is a replication fork. 00:13:17.14 Over here is another replication fork, and the 00:13:19.09 origin of DNA replication would have been here 00:13:22.08 and up here, and this is now duplicated DNA 00:13:25.16 And these forks will travel apart, making completely replicated DNA. 00:13:31.00 In a eukaryotic cell the chromosomes are not circular. 00:13:34.05 They are linear, but their linearity doesn't make the problem easy 00:13:39.25 and I mention that the DNA is held together as it is replicated. 00:13:44.12 So here is a replication fork, 00:13:46.23 here are sister chromatids which have been produced 00:13:49.29 by replication, and this is a complex called the cohesin complex, 00:13:54.25 which in some way fastens these sister chromatids, 00:13:58.13 as they're called, together so that as the DNA wraps on 00:14:02.22 the nucleosome core particles, which is the first stage of condensation, 00:14:08.11 the sister chromatin, which is the name for the material that is DNA plus protein 00:14:13.03 is held together, and these sisters are associated. 00:14:16.27 Exactly how the cohesin complex does this is 00:14:20.16 still not fully understood, but this diagrammatic 00:14:23.29 representation of it surrounding the two is a plausible way to think about 00:14:28.10 how it could link sister chromatids together. 00:14:30.26 Once DNA replication is complete, condensation will occur, 00:14:35.25 and here are multiple stages of the condensation diagrammed 00:14:38.20 in a textbook form. 00:14:40.21 The condensation is going to give us, in most cells, thousands of fold 00:14:46.06 decrease in length. 00:14:47.25 In some cells, it is not so much. So, as is common in biology, 00:14:51.27 there's a lot of variability and you have to account for that 00:14:55.15 as you are thinking about trying to understand a process in a simple term. 00:15:00.08 But, all eukaryotic cells use this packing on nucleosomes 00:15:04.12 and the nucleosomes then pack together 00:15:06.24 to form this fiber which looks like about 30 nanometers in diameter 00:15:12.12 when it is seen in an electron microscope and then 00:15:14.19 this folds and loops, and then the loops condense, 00:15:17.08 and finally it comes to the chromosome, 00:15:20.18 which name, of course, means the colored body. 00:15:23.11 And that gets that name because once it is condensed sufficiently 00:15:28.08 you can see these strands of DNA in the light microscope 00:15:32.07 with sufficient, good resolution and stains. 00:15:36.00 And chromosomes were recognized as such even in the 19th century. 00:15:40.03 Now there's still a lot of work going on to try to understand this process 00:15:44.20 and proteins have been discovered that were thought at first 00:15:48.10 to be extremely important for it, for example a protein 00:15:51.02 called cohesin, I'm sorry, condensin, which is involved 00:15:55.03 in making the chromosomes become more condensed. 00:15:58.10 But it turns out that that protein is not necessary 00:16:02.15 for the condensation process, it probably depends instead on 00:16:07.22 a combination of post-translational modifications of the proteins that 00:16:11.07 associate with the DNA in order to compact 00:16:14.05 the chromatin by changing their charge 00:16:16.21 and probably changing the proteins with which they associate. 00:16:19.13 So this condensation will bring us to the time in which we can enter division 00:16:24.27 itself, and this is a still frame from a movie 00:16:28.10 taken by my friend Jeremy Pickett-Heaps, of a newt cell in the process of cell division. 00:16:33.27 The nuclear envelope is still intact, and you see the condensed chromosomes 00:16:38.15 here within the nucleus, and as I run the movie what you'll see 00:16:41.29 is that the nuclear envelope breaks down and something now effects the chromosome behavior. 00:16:49.03 Chromosomes appear to be moving and becoming organized. 00:16:52.02 And this is of course the process of moving towards 00:16:54.27 metaphase. It is the stage called pro-metaphase 00:16:59.10 and the process of moving chromosomes to the metaphase plate 00:17:02.27 is called congression. So they are gradually getting lined up, but it is clear 00:17:08.01 that there are also renegades that don't get in line in time 00:17:12.18 and some of them will even depart and then go back, but eventually anaphase will start. 00:17:17.20 Anaphase is this process of ordered segregation followed by 00:17:21.25 cytokinesis where the cell pinches in the middle in animals cells. 00:17:26.00 In plant cells, you build a wall between the two daughter cells 00:17:30.12 instead, but what this gives rise to then is two independent nuclei, 00:17:34.24 each with a complete fabric of chromosomes, 00:17:37.26 and they are now divided into two distinct cells. 00:17:41.23 Now in the movie that I just showed you, you saw chromosomes 00:17:45.22 and their behavior, but we didn't see anything about the mitotic spindle, 00:17:49.07 which I said in the diagram awhile ago 00:17:51.29 is responsible for this event. The mitotic spindle 00:17:54.26 can be visualized in living cells in a variety of ways. 00:17:59.00 Historically, the most important was the use of some optics that involved polarized light. 00:18:03.22 And a number of people, principally Shinya Inoue have 00:18:08.19 been responsible for taking advantage of polarized light 00:18:11.23 in order to visualize the machinery for chromosome movement as it acts. 00:18:16.19 This is a movie that I am going to show you that is taken 00:18:18.23 with a brand new kind of polarization microscope, which 00:18:23.11 gives a brightness reflecting the way in which polarized light is interacting 00:18:29.08 with the cell in such a fashion that it doesn't depend on the orientation 00:18:32.09 of the object relative to the plane of polarization. 00:18:36.10 And this turns out to be important for getting a clear image, and now 00:18:40.02 brightness is the polarization optical image 00:18:43.15 of the spindle. This is the same cell type that you are seeing, and there's the same anaphase. 00:18:48.15 And you can see that the spindle shortens as the chromosomes 00:18:51.17 draw to the poles, but the whole structure also elongates 00:18:55.29 in order to give you the greater separation of the chromosomes that we saw 00:18:59.18 at the end of that phase microscopy movie. 00:19:03.14 Here's a diagrammatic representation, although this is actually an electron micrograph 00:19:08.19 taken of a mammalian cell in culture, 00:19:12.10 which was fixed and then stained for tubulin 00:19:15.27 using colloidal gold to bring out the microtubules 00:19:18.28 that are here. The chromosomes are evident just from their own binding of stain. 00:19:23.23 And here I've diagrammed with my arrows where the poles of the spindle are, 00:19:28.10 where the chromosomes make contact with 00:19:31.02 fibers that come from the poles, 00:19:33.27 and the special region on the chromosome 00:19:36.25 to which these fibers attach is called the kinetochore. 00:19:40.05 This comes from the Greek root meaning movement. 00:19:44.10 Out here we have fibers which extend radiating out from the poles. 00:19:48.27 These are often called the astral rays, and they are common in many animal cells, 00:19:53.04 but they are by no means universally found. 00:19:55.10 They appear to be part of the process that will center the spindle within the cell 00:20:00.12 and maybe part of the elongation of the spindle process, but they are not 00:20:03.25 essential for organizing and segregating chromosomes. 00:20:06.20 This micrograph gives us an overview of 00:20:10.13 what the spindle looks like, but we can look more deeply into the spindle 00:20:15.00 by means of higher resolution electron microscopy. 00:20:18.08 here cutting thin sections to see the kinetochore 00:20:21.17 as a layer of proteinaceous material which is stuck onto the underlying 00:20:27.23 chromatin, which is here. 00:20:29.21 Microtubules are coming out from this kinetochore 00:20:34.04 and this attachment is a key part of forming a functional spindle. 00:20:40.02 The spindle in many cells grows from a structure called the centrosome 00:20:45.03 The centrosome contains two centrioles, 00:20:49.04 so called in many animal cells, and some pericentriolar material. 00:20:53.25 This includes a special isoform of tubulin called gamma-tubulin 00:20:57.18 which is held in place by a series of other proteins, 00:21:01.19 long fibers, like pericentrin, which are holding the gamma tubulin in place 00:21:06.13 making a series of microtubule nucleating sites 00:21:09.23 from which tubulin can polymerize 00:21:12.14 to make the microtubules of the spindle. 00:21:14.25 This is what a cell looks like as it goes into mitosis, 00:21:19.27 again an electron micrograph with the chromosomes shown very dark 00:21:23.14 the spindle poles at the ends of the spindle and you can see 00:21:26.29 that the distance between the chromosomes and the 00:21:29.12 poles has begun to shorten. This shortening is called anaphase A. 00:21:34.23 And it's an essential part of the segregation of chromosome process. 00:21:39.02 The spindle elongation is called anaphase B. 00:21:42.13 And it leaves behind this interzone in the middle which appears in this micrograph to be 00:21:48.00 essentially empty but this is simply because those microtubules are not being stained 00:21:53.16 in such a way that they show up here. There are actually microtubules 00:21:56.14 there. And I can show you this, again with polarized light 00:22:00.12 looking at a meiosis spindle, Meiosis II, which is mechanically very much like 00:22:06.18 a normal mitosis. And this is in the spindles of a crane fly, which is 00:22:13.05 a particularly photogenic object. 00:22:15.24 So again with polarized light we'll see here the two cells that are the products of the 00:22:21.04 meiosis I and they are now each going to form their own mitotic 00:22:25.16 spindle, which will then organize the chromosomes 00:22:28.26 and you now see the chromosomes as ghosts, 00:22:31.00 on the spindle equator. Anaphase will start 00:22:34.13 and the chromosomes are now going through Anaphase A and Anaphase B 00:22:39.04 with the birefringence in the middle of the spindle 00:22:42.07 giving us an indication of the amount of structure 00:22:45.01 that's there as these four spermatids are produced from the second meiotic division. 00:22:53.00 Now, electron microscopy is used, has been used to try to 00:22:58.15 understand the structure of the microtubules as they are arranged in the mitotic spindle. 00:23:03.02 And this is from some old work in our lab in which we took a variety 00:23:07.12 of cells from Dictyostelium, one in metaphase, one in early 00:23:12.17 anaphase, and then later and later and later anaphase, 00:23:15.15 looking at this mid region where that previous electron micrograph didn't 00:23:19.23 really show any indication of microtubules. But this is actually a count of the number of 00:23:24.27 microtubules as a function of position along the spindle axis, 00:23:28.08 and you can see that there are a large number 00:23:30.07 of tubules in that middle region. 00:23:33.24 This is a diagram that we built on the basis of electron microscopy 00:23:38.18 of the kind I've been showing you and a method that I'm not showing here, 00:23:42.17 which is a technique that revealed the directionality of individual microtubules. 00:23:48.13 Every microtubule is a polar structure 00:23:51.10 because it is assembled from an asymmetric protein 00:23:53.23 molecule and each protein adds on in a head to tail fashion 00:23:57.22 so each microtubule is a vector. 00:24:00.24 The two ends of every microtubule are different. One end is called the plus end, 00:24:05.08 the other the minus. The plus gets it name because that's the end 00:24:09.21 that experimentally one can see microtubules grow and shrink faster at the plus end. 00:24:14.21 And the spindle is designed in such a way that 00:24:17.16 the centrosomes at the poles nucleate the microtubules 00:24:21.19 and the microtubules then grow outwards with their plus ends distal 00:24:26.09 and plus ends also are the ends that interact with the kinetochore 00:24:30.17 and making a structure which is in effect two fold symmetric. 00:24:35.00 This two fold symmetry, that is that you could either look at it like this or like that 00:24:41.02 and it's going to be the same, the two fold symmetry 00:24:44.14 is maintained as the chromosomes are segregated 00:24:46.24 giving rise to nuclei at either end of the spindle, and again 00:24:50.14 interdigitating microtubules left in the middle in the interzone with 00:24:55.08 their plus ends gathered in the central region. 00:24:58.17 This structure towards the end of an animal cell division 00:25:02.04 is called the midbody. 00:25:06.10 Spindles from different organisms show some elements of similarity 00:25:10.16 but also some elements of real diversity. 00:25:13.14 What I'm showing up here in this top diagram 00:25:17.10 is an accurate reconstruction done by electron tomography 00:25:20.28 of all the microtubules that are present in the spindle of a budding yeast. 00:25:25.05 This work was done by Eileen O'Toole in our lab in Boulder 00:25:28.23 and you can see the nuclear envelope that is still 00:25:32.10 surrounding this nucleus and this spindle 00:25:36.17 because the budding yeast, like many simple cells, 00:25:40.05 is one which keeps its nuclear envelope more or less intact, 00:25:44.08 brings protein for mitosis into the nucleus, and then builds a spindle right in the nucleus. 00:25:50.10 A system which is quite different from what we have just seen 00:25:54.11 looking in vertebrate cells. 00:25:56.06 But this spindle is different in a number of ways from, for example, 00:26:00.29 a mammalian cell. And this is some more of Eileen's work, 00:26:03.26 where she's in the process of doing a reconstruction 00:26:06.06 of a human cell which is now much longer in extent 00:26:13.06 and just how much longer is evident if I show you 00:26:17.08 in the upper right hand corner here a yeast spindle 00:26:20.26 which you can just barely see up in that corner 00:26:23.17 that is a one micrometer yeast spindle 00:26:25.11 shown in nice, correct proportion to the mammalian spindle here. 00:26:30.25 So there are very big differences in scale 00:26:34.11 as well as is the nucleus intact or not. 00:26:38.04 One can find even more indications of variety 00:26:42.15 as you look around in mitosis. So this is a cell 00:26:46.06 which is called Barbulanympha. It's a flagellate that lives as a sort of symbiont 00:26:52.25 in the hindgut of a wood feeding roach 00:26:55.13 where it participates in the digestion of the cellulose which 00:26:59.12 is necessary for that roach to get the energy that it needs. 00:27:02.10 And this is a huge cell. And you can see here with this ten micrometer marker 00:27:06.07 that it's very big relative to a mammalian cell. 00:27:09.10 These are centrosomes which are still in the cytoplasm. 00:27:13.10 The nucleus is intact, and as this cell goes into mitosis, 00:27:17.06 remarkably, the spindle stays in the cytoplasm. 00:27:23.00 The nuclear envelope does not break down, and yet the chromosomes associate 00:27:26.27 with the nuclear envelope and attach to spindle fibers 00:27:30.23 that were developed in the cytoplasm right through the nuclear envelope. 00:27:34.16 So you can see that there is considerable variety not only in the size 00:27:40.01 of the spindle, but in the sort of topography, 00:27:43.21 the way in which it goes about setting up the division process. 00:27:46.21 When you have this kind of diversity, variability in form and function, 00:27:52.20 how do you understand it? 00:27:54.14 And the answer in general in biology is look for things that are consistent 00:27:59.14 between all the various different structures with which you deal, 00:28:02.25 but also you want to use variation whenever you can 00:28:06.21 in order to take advantage of the strength of one system 00:28:09.25 versus another. And that will come back as a theme 00:28:12.14 in my section on experiment in the next lecture. 00:28:14.24 But the things that are consistent in spindles 00:28:18.14 are that all spindles use microtubules as their major fibrous component. 00:28:23.04 All spindles are organized in such a way that the microtubules 00:28:26.17 have their plus ends pointing away from the spindle pole and pointing into the chromosomes. 00:28:32.08 They build a structure that is essentially two fold in its symmetry 00:28:37.11 and is therefore anticipating the functional symmetry 00:28:40.12 which we'll see when anaphase begins. 00:28:44.00 All mitotic spindles have the ability to attach to chromosomes, 00:28:48.26 and this attachment to chromosomes is an essential feature 00:28:52.25 of being able to pull on the chromosomes 00:28:54.15 and effect their segregation. When segregation is occurring, anaphase 00:28:59.17 always incurs the separation of sister chromatids 00:29:04.04 followed by their motion in opposite directions. 00:29:07.09 So what we have are a couple of fundamental principles 00:29:10.25 of how mitosis is going to work. And in the next lecture 00:29:14.11 we'll look inside the cell to see if we can understand 00:29:18.02 how this remarkable machine really works.