Session 7: Cell Division

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.

00:00:09.24 Hello, my name is Dick McIntosh, and I am a professor
00:00:12.29 of Cell Biology at the University of Colorado
00:00:15.11 and this is the second of three lectures in which I am talking about
00:00:18.05 chromosome movement and cell division. In the first lecture,
00:00:22.20 we talked about background material,
00:00:24.11 that is the cells getting ready for division;
00:00:26.22 now we are going to look at experiments and the ways in which
00:00:30.04 they have informed us about the machinery that does
00:00:33.12 the division process. In the previous lecture I emphasized
00:00:37.23 the complexity of cells and the difficulties
00:00:40.27 that one encounters because they are so small.
00:00:42.22 This pair of factors really has a huge impact
00:00:47.22 on both the work that people have done to try to understand cell division
00:00:52.18 and on the results that many excellent scientists have gotten,
00:00:56.29 as they have pursued this challenging problem.
00:01:00.02 So what you'll hear about in this lecture is a range of
00:01:03.19 methods of experimentation, approaches that people have used to try to
00:01:09.04 perturb the spindle in informative ways.
00:01:11.18 And some of them included genetic approaches,
00:01:14.17 some have included mechanical approaches,
00:01:16.25 and some are biochemical, some are immunological,
00:01:20.09 using antibodies that can block functions,
00:01:22.14 and some are pharmacological, using drugs.
00:01:24.15 And I'll show examples of a variety of these things,
00:01:27.17 in order to give you a sense of the experimental richness
00:01:30.29 that is available to us in trying to understand this complicated process.
00:01:34.17 The difficulty is that experiments have been done on different organisms
00:01:40.06 and one cannot always compare information that comes from these different organisms
00:01:45.29 in a literal way because the mitotic processes in different cells may not be identical.
00:01:50.25 Further than that, they may respond differently to a
00:01:54.20 particular pharmaceutical agent or to a particular mutation,
00:01:58.00 so we have the complexity that is brought into this process of
00:02:01.20 biological variability. Of course there's the
00:02:04.08 biological variability of the experimenter as well.
00:02:07.06 So, by no means can I tell you at the end of this lecture how mitosis works,
00:02:13.11 but what we'll be able to do is see the relative importance of several aspects
00:02:17.22 of spindle function and the ways in which
00:02:19.29 they have impact on chromosomal movement.
00:02:22.11 So the jobs that the cell has to do in order to segregate chromosomes
00:02:27.13 accurately is to make the mitotic spindle.
00:02:30.27 It has to attach the chromosomes to the spindle
00:02:33.26 and organize them and then segregate them into the two distinct sets.
00:02:39.04 The approaches we are going to take in this lecture to these different problems are
00:02:44.13 quite different. The first one is simply to bow out because Ron Vale
00:02:49.01 has done a very nice lecture on spindle formation showing
00:02:53.07 some beautiful work from his lab that illuminates aspects
00:02:57.07 of this complicated phenomenon. So I am really not going to deal with it at all here.
00:03:01.28 On the other hand we will focus on the mechanical properties of the spindle in order to see
00:03:07.27 how they affect chromosome attachment and chromosome segregation.
00:03:12.23 Now I am going to start with spindle properties
00:03:15.27 and then go on to the elongation of the spindle
00:03:20.06 because that's a simpler problem than
00:03:22.16 the whole business of attaching chromosomes to the spindle and organizing
00:03:26.10 their segregation, but we'll get there.
00:03:28.24 I have included this beautiful slide, a text book slide,
00:03:33.18 the work of Bill Earnshaw using immuno-fluorescence with
00:03:36.03 red staining to show the microtubules and blue staining
00:03:39.04 to show the chromosomes because it displays
00:03:42.02 very beautifully the overall pattern of spindle formation
00:03:46.08 and function in a wide variety of vertebrate cells.
00:03:50.23 We are going to form this metaphase structure
00:03:53.20 by means of attaching the chromosomes
00:03:56.14 to the microtubules that will come out of the cytoplasm
00:04:00.10 into the nucleoplasm and then organize
00:04:02.18 the chromosomes to produce this structure.
00:04:05.02 Anaphase will occur, in two stages: Anaphase A,
00:04:09.06 which is that shortening of chromosomes to poles.
00:04:11.18 Anaphase B then is the elongation and then finally cytokinesis will come
00:04:17.02 and pinch that bundle of microtubules down
00:04:20.11 into a single shaft that I have called the mid body.
00:04:23.08 This process is fairly well conserved in its overview.
00:04:28.24 The details vary, but we can take this as the general process for mitotic action.
00:04:34.18 And I showed this slide in the previous lecture to emphasize
00:04:38.18 the simple symmetry of the spindle once it is formed.
00:04:42.07 The spindle is bipolar because it grows from two poles
00:04:46.10 and these microtubules interact with one another,
00:04:49.04 and some of them interact with the chromosomes
00:04:51.06 setting aside the particular microtubules
00:04:55.02 that are called kinetochore microtubules,
00:04:57.01 because those are the ones that interact with this
00:04:59.23 chromosomal specialization, the kinetochore.
00:05:02.15 When anaphase starts there is a severing
00:05:05.11 of the connection between sister chromatids,
00:05:08.04 and then the chromatids move apart and we are left behind
00:05:12.00 with this interzone spindle which can elongate in order
00:05:15.13 to increase the distance between the poles and achieve anaphase B.
00:05:23.10 Now the first question I want to address is that of
00:05:26.25 how do those two half spindles interact with one another
00:05:31.28 to form the two fold symmetric structure?
00:05:34.15 Because if you imagine structures that are simply forming microtubules
00:05:39.08 like a radial array that might grow out of a centrosome,
00:05:41.20 it isn't clear that those microtubules
00:05:44.23 would interact, but they do. And they do as the result of several protein factors.
00:05:50.16 One factor that has now been identified, actually comparatively recently,
00:05:54.09 is a microtubule binding protein called Ase1.
00:05:58.29 And Ase1 has the property that it is localized at the spindle here
00:06:02.09 this is showing you work from a fission yeast cell, which is this elongate structure.
00:06:07.24 Here are the microtubules shown in red,
00:06:10.18 and then here is this Ase1 protein shown in green
00:06:14.08 and the two of them overlapping at the bottom.
00:06:17.09 Ase1 is concentrated in the region where those two half spindles are interdigitating
00:06:22.27 and genetic work in pombe has shown that when
00:06:26.03 you delete the Ase1 gene, the spindle frequently falls apart
00:06:30.11 into two distinct halves, so this is a mechanical agent
00:06:35.03 that is helping to hold together interdigitating microtubules
00:06:38.13 at the middle of the spindle.
00:06:43.05 Ase1 is not the only factor that is important for this process however,
00:06:46.29 enzymes are also required to establish spindle bipolarity
00:06:51.03 and the central one in many cell types is this unusual looking molecule,
00:06:56.22 which is four single polypeptides that are identical, so it is a homo-tetramer,
00:07:02.24 and it assembles to form a bipolar structure in which there are two heads at each end.
00:07:08.28 And each of these heads is a motor enzyme
00:07:12.07 so this homo-tetrameric molecule of the kinesin family
00:07:18.07 is able to bind to microtubules and one form of binding is shown here,
00:07:23.16 where the microtubules are what we say is anti-parallel,
00:07:27.04 that is the plus end is here on this microtubule and pointing in the opposite direction there.
00:07:32.16 This kinesin-5 is a plus end directed motor, so it walks
00:07:38.23 towards the plus ends of the microtubules
00:07:40.21 and that means that it is going to rearrange the microtubules over time.
00:07:45.01 When it's interacting between two
00:07:47.23 parallel microtubules on the other hand, if it walks towards their plus end,
00:07:52.00 it doesn't cause the microtubules to slide,
00:07:54.00 but instead it moves relative to the microtubules,
00:07:57.03 up towards their plus ends.
00:07:59.00 And down at the bottom, is a diagram of a pole
00:08:02.01 and then an organized spindle, which represent the results
00:08:06.01 of the action of this kinesin-like protein
00:08:08.29 as it interacts with the microtubules that have come from two poles
00:08:12.22 and it pulls them together to form a bipolar structure in which there is
00:08:16.24 only a limited amount of overlap near the middle of the spindle.
00:08:25.09 The importance of this kinesin-5 has been demonstrated in several ways.
00:08:32.10 Genetically if you remove the gene for this motor
00:08:36.05 from yeast for example, what you find
00:08:40.07 is that the bipolar spindle simply will not form,
00:08:43.10 but that could be an indirect effect of some kind.
00:08:45.24 You could argue that without this motor you don't transport
00:08:48.17 some essential component to the middle of the spindle
00:08:51.21 and that is what is doing the linking.
00:08:53.23 A different kind of evidence
00:08:55.25 has been provided by pharmacology in which a number of different groups
00:08:59.11 have sought small molecules that will interact specifically with this kinesin-5 and inactivate it.
00:09:05.22 And one of the early ones is called Monastrol.
00:09:08.28 There's been quite a lot of experimentation done with it because
00:09:11.27 it seems to be quite specific when it acts in mammalian cells.
00:09:15.09 And what one finds is that a monastrol treated cell
00:09:19.17 going into mitosis forms a monopolar spindle.
00:09:23.07 There are two centrosomes in the middle here, but they can't separate.
00:09:27.07 They can't form independent units of astral microtubules which can interact because without
00:09:34.11 that motor there to push things apart the spindle just never forms.
00:09:39.22 So the activity of kinesin-5 is essential for forming a bipolar spindle.
00:09:45.06 This is a reversible effect, because if you take out the drug,
00:09:49.07 the spindle will form and function in anaphase perfectly well.
00:09:52.01 And what you can see in this pair of images
00:09:55.01 is that if you take the monastrol poisoned spindles, which are monopolar,
00:10:00.05 and use a reagent that will tend to dissolve the microtubules,
00:10:04.08 calcium ions will do this,
00:10:06.02 then what happens is you are left simply with bundles of microtubules
00:10:10.10 that connect directly to kinetochores.
00:10:13.19 So, what we have in this structure is evidence that monastrol
00:10:18.07 is very important for the interdigitation and the interaction of the microtubules
00:10:22.19 that are coming from the two poles.
00:10:25.25 Surprisingly chromosomes are not required to have a bipolar spindle,
00:10:31.12 and this is a really rather remarkable genre of experimentation involving
00:10:35.24 micromanipulation, started really and developed
00:10:39.10 most fully by Bruce Nicklas, but his student Dahong Zhang
00:10:43.05 has done a wonderful experiment here in which he has used a micro needle
00:10:47.20 to reach into the cell and remove the chromosomes,
00:10:50.24 pulling them off into another region.
00:10:53.19 So we have a spindle forming with no chromosomes on it.
00:10:57.01 It is being seen in polarized light here, and here we get a change from bright to dark
00:11:02.29 as a result of this being a linearly polarized microscope
00:11:07.02 and this structure here is similar to what is shown down here with
00:11:11.07 antibodies to tubulin revealing where the microtubules are.
00:11:13.26 This structure is perfectly capable of going ahead
00:11:17.24 and helping the cell set up a cleavage furrow
00:11:21.12 even with no chromosomes and it retains its bipolarity
00:11:24.10 quite nicely as it functions with the chromosomes gone.
00:11:28.01 Now, when chromosomes are present the spindle is mechanically somewhat different,
00:11:35.27 and I am going farther afield in terms of biology
00:11:38.23 here in order to make this point. This is the spindle of a diatom,
00:11:43.06 which is a kind of alga, and it has the virtue that the interpolar spindle
00:11:48.29 is sufficiently well organized that it gives rise to a very birefringent object
00:11:56.02 that shows up in the polarizing microscope.
00:11:58.05 And Jeremy Pickett-Heaps and his students
00:12:00.27 used polarization optics to visualize this spindle and then
00:12:04.13 a micro-beam of ultraviolet light in order to irradiate a small portion of the spindle,
00:12:10.07 and they destroyed the microtubules in that area
00:12:13.11 with this perturbation. And what you can see in the row of images
00:12:17.05 across the bottom is that when the spindle is no longer symmetric in its strength,
00:12:22.10 it bends inward, revealing the fact that the poles are being pushed inwards
00:12:27.05 towards one another by the mitotic structure.
00:12:30.16 The chromosomes are apparently being pulled towards
00:12:33.28 the poles and they are pulling inwards on the poles
00:12:37.13 as this mechanical equilibrium is set up during the mitotic process.
00:12:42.10 Well, what is pushing out in order to prevent the poles from just collapsing?
00:12:48.06 And the answer probably is in large part this same kinesin-5
00:12:52.08 that we talked about before. Here are immuno-localization images
00:12:56.10 in which we can see kinesin-5 and tubulin and then the superposition
00:13:01.11 of those two shown in different colors.
00:13:04.03 And you can see here that kinesin-5 is plentiful in the spindle
00:13:08.22 and it is found in this mid-region
00:13:11.13 in the spindle where there are not even quite so many microtubules
00:13:14.12 and in that region its function of crosslinking anti-parallel microtubules
00:13:20.07 and walking towards the plus ends of the microtubules
00:13:23.02 which will tend to push apart is going to
00:13:25.00 provide a force that will tend to keep the poles from collapsing.
00:13:29.07 But as is so often is true in biology, things are not that simple.
00:13:33.26 Kinesin-5 is not the only motor in the spindle;
00:13:36.11 there is also a different kinesin, and in this case it is called kinesin-14.
00:13:42.07 which has the opposite polarity of motion.
00:13:45.05 it walks towards the minus ends and so in this image here
00:13:49.18 what you can see is that kinesin-14, represented in green,
00:13:55.19 is concentrated in the middle of the spindle
00:13:58.18 tubulin represented by the red staining, which is giving us purple towards the poles
00:14:04.24 is more concentrated towards the poles,
00:14:07.25 and so this intermediate region, the interzone,
00:14:11.18 even at metaphase, is a region where we find kinesin-14
00:14:15.22 that pushes the microtubules towards the middle
00:14:19.09 of the spindle and kinesin-5 which pushes them away.
00:14:22.07 Suggesting some kind of a dynamic equilibrium between them.
00:14:25.27 This is diagrammed here as a balance of forces
00:14:29.13 in which kinesin-5 is pushing outwards as it walks
00:14:34.04 towards the plus ends of the microtubules and the kinesin-14 is pulling inwards
00:14:39.28 as it walks towards the minus ends of the microtubules
00:14:42.13 and evidence for this kind of balance comes from genetic experiments
00:14:47.19 where if you delete the kinesin-5 then the spindle will tend to collapse.
00:14:51.23 So we have a combination here of the mechanics
00:14:57.05 that is offered by the stability of the microtubules themselves
00:15:00.18 and motors in the middle that are pushing and pulling,
00:15:03.08 so we can regulate quite carefully what is going to happen
00:15:06.17 in this zone of overlap. I've often thought about this a little bit the way
00:15:10.22 of how you might think about how you do fine motor control.
00:15:13.16 You want to be able to push in both directions.
00:15:15.19 So if you hold something between thumb and fingers
00:15:18.26 and now you can manipulate it quite precisely like the violin bow or something.
00:15:22.28 And here the spindle is manipulating the interzone
00:15:26.21 microtubules by being able to both push and pull on them.
00:15:29.23 at the same time. But this is not all that is going on in this spindle,
00:15:34.28 there are also the dynamics of the microtubules themselves.
00:15:38.23 Microtubules, of course, can polymerize and de-polymerize and this cycle
00:15:44.00 has been well described by many people in many labs.
00:15:47.28 And I am not going to dwell on it here.
00:15:49.11 But polymerization involves the assembly of tubulin
00:15:53.09 that has GTP bound to it. The GTP is hydrolyzed,
00:15:57.01 and then when disassembly occurs these microtubule strands,
00:16:03.03 so called protofilaments seem to bend during the course of the disassembly process.
00:16:07.09 This kind of dynamics is going on in the spindle all the time,
00:16:11.24 and there is good evidence for this: evidence has come from photobleaching,
00:16:16.24 where you can see the spindle microtubules turn over quickly,
00:16:19.25 but the most remarkable evidence for it has come from using a fluorescent tubulin
00:16:24.17 in order to mark individual microtubules in the cell
00:16:27.20 and take advantage of very sensitive cameras
00:16:30.26 to be able to see this fluorescence even when there is so little there
00:16:34.03 that a microtubule is not uniformly labeled,
00:16:37.06 but it is heterogeneously labeled or it looks like speckles.
00:16:40.16 And this is called speckle imaging and it's been used by a number of investigators,
00:16:45.25 having been invented by Ted Salmon and Clare Waterman
00:16:49.01 as a way of looking at microtubule dynamics in living cells.
00:16:53.15 And here I am showing you some spindles that were imaged
00:16:56.12 with this method showing that the microtubules
00:16:59.08 of the spindle are continuously moving towards the pole
00:17:02.04 in both directions as if kinesin-5 is pushing them outwards from that zone of overlap
00:17:09.04 in the middle. but if that were true, the spindle should be elongating and it's not.
00:17:14.15 suggesting that there's control on the microtubule dynamics
00:17:18.13 and this dynamics comes from yet another microtubule motor, a kinesin-13
00:17:25.05 It has the behavior that it can help promote disassembly of microtubules,
00:17:30.26 one kinesin-13, anyway, is concentrated at the spindle poles.
00:17:36.19 and that means that it can help to chew up the microtubules
00:17:40.06 as they are pushed towards the pole,
00:17:42.06 allowing the spindle to treadmill away from the center without getting longer.
00:17:47.28 And indeed in this work from Sharp, you can see that as the microtubules
00:17:53.01 are being followed with speckle imaging
00:17:55.04 when the kinesin-13 has been inactivated by antibody injection
00:18:00.16 the motion towards the poles reflected here by this movement outwards
00:18:06.27 because this is a time axis and this is a space axis
00:18:10.24 and the slope of these lines reflects how fast these speckles are moving
00:18:16.04 The speckles are moving much more slowly when we impede
00:18:19.21 the activity of this disassembly motor at the poles.
00:18:22.29 So we not only have motors functioning as mechanical entities pushing,
00:18:28.01 we have motors functioning in the dynamics of spindle microtubules.
00:18:34.06 This can all be assembled in a diagram and I have taken
00:18:38.04 this very nice diagram from the Pollard and Earnshaw textbook
00:18:41.27 showing microtubules attached to chromosomes in the middle,
00:18:45.20 the addition of subunits in this region here,
00:18:49.18 and the motion of the microtubules towards the pole
00:18:51.21 in a process which Tim Mitchison called "flux".
00:18:54.17 We also have the overlapping microtubules in the middle here,
00:18:58.20 we are adding subunits on either side of this overlap region at the plus ends
00:19:04.22 of the microtubules and pushing the microtubules towards
00:19:08.03 the pole using the kinesin-5, but balancing
00:19:11.04 that action with the kinesin that is working in the opposite direction in the middle.
00:19:16.11 So this is a complicated structure that is sliding microtubules
00:19:20.21 towards the pole all the time that it appears simply to be sitting there in metaphase.
00:19:26.22 Does this really explain chromosome motion?
00:19:30.12 You can think of it in the way that if all the microtubules
00:19:33.17 in a metaphase cell were sitting there in flux, anaphase A could happen simply by
00:19:40.12 allowing the separation of the sister chromatids,
00:19:43.06 and then allowing each of the sister chromatids
00:19:46.08 to join the flux and go to the poles. Anaphase B could happen simply by
00:19:50.24 turning off the disassembly that is going on at the poles,
00:19:54.11 and this summary may be part of how mitosis really works.
00:19:58.25 At least in some cells, but we have biological variability to deal with,
00:20:04.28 and here now I am going back to fission yeast.
00:20:08.00 and we are taking advantage of the ability to perturb fluorescence in a cell.
00:20:13.21 The fluorescence here is coming from tubulin
00:20:17.05 which is marked with a fluorescent dye, and in this live cell
00:20:20.19 you are seeing a series of time frames here of a normal spindle as it elongates
00:20:26.07 Here we have a spindle and we are photobleaching the fluorescence in the middle
00:20:30.27 that means we use a bright light in order to kill the fluorescence,
00:20:35.02 but the microtubules are still there.
00:20:37.04 And what you can see is that the fluorescence comes back,
00:20:41.09 on the other hand if we go a little later in mitosis,
00:20:43.11 and we photobleach in the middle, what you now see is that
00:20:47.03 that spot divides in two, and separates and moves towards the poles .
00:20:51.17 This is just what you would expect if you had those overlapping tubules in the middle
00:20:57.00 and they were sliding apart and you put a mark on them, those marks would move apart.
00:21:01.15 But we are not seeing flux. This is Anaphase B and it is the migration of the poles apart.
00:21:09.03 and there is no indication of dissolving the microtubules at the poles in this cell.
00:21:15.13 So flux does not seem to be a universal, and yet these cells
00:21:20.05 and many others where flux does not appear
00:21:22.07 to occur, divide chromosomes perfectly well.
00:21:25.07 So flux may be important, but it can't be the whole answer.
00:21:28.04 There is even more to the mechanics of this because we learned a long time ago
00:21:33.13 from some work that was done with beautiful micro-irradiation
00:21:37.12 again with ultraviolet light, and this has now been confirmed by laser irradiation
00:21:41.25 with some more recent experiments. In a number of fungi, if you have an un-irradiated cell
00:21:48.11 and you measure the rate of spindle elongation you get some value
00:21:52.27 and you would imagine now, if we irradiated the middle
00:21:56.00 and kill this region where I've been telling you kinesin-5 has been acting,
00:22:00.06 what would happen then is that the spindle then would either collapse
00:22:03.25 or would no longer elongate, or it would elongate more slowly.
00:22:07.05 But what happens in fact is that it elongates faster.
00:22:10.13 We have a situation here where the experimental evidence suggests
00:22:15.02 that that central bar in the spindle is helping to direct
00:22:19.18 chromosome movement, and helping to regulate it,
00:22:22.09 but it is not the driving force in these cells.
00:22:25.05 And down below are important control experiments where
00:22:28.05 you miss the spindle in the middle and have essentially no effect on the rate or the velocity
00:22:33.14 and irradiate outside the spindle and again. So this looks like a very
00:22:37.19 reliable result, and indeed as deeper work has gone on in the laboratory
00:22:42.13 of Gero Steinberg recently using genetics as well,
00:22:45.24 he has been able to show that the probable motor
00:22:49.16 for this in the cell he has been studying is a dynein
00:22:53.22 which is somehow anchored in the cortex
00:22:56.14 of the cell and is able to walk towards the minus ends of the microtubule
00:23:00.15 that grow out of astral arrays and help to pull the poles apart.
00:23:06.11 So it looks as if spindle elongation in at least some cells,
00:23:10.01 and this is probably true of fungi and others,
00:23:12.13 is a front wheel drive, not a back wheel drive.
00:23:15.17 Is the back wheel drive important?
00:23:18.05 Yes, because if the two spindles went off in different directions like this,
00:23:22.29 you might imagine that what would happen then
00:23:25.25 is that when cytokinesis occurred the two nuclei would wind up in the same daughter cell.
00:23:30.15 so it is important that the direction of motion be in opposite directions,
00:23:36.25 so that we can get these daughter nuclei into very distant regions in the cell.
00:23:43.12 So the pulling is mechanically important,
00:23:46.00 but for sure this middle region is important
00:23:48.24 in guiding and controlling even in those cells where it doesn't seem to be
00:23:53.06 the major driving force for the motility.
00:23:55.29 So now we are ready to start talking about
00:23:58.17 interacting with the chromosomes and how does the spindle
00:24:01.17 bind them. Over on the left here are two stages
00:24:06.12 from that fluorescence pair that I showed at the very beginning of this lecture
00:24:10.03 and as you can see we start out with the microtubules in the spindle that is forming
00:24:15.10 and the chromosomes scattered all around.
00:24:17.07 But they come to this very ordered arrangement of metaphase.
00:24:21.06 Here is an electron micrograph again from the laboratory of
00:24:25.15 Jeremy Pickett-Heaps looking at the early stages
00:24:29.04 of chromosome attachment to the spindle.
00:24:31.09 The important features here are that these are two chromatids.
00:24:35.19 You can imagine their arms extending
00:24:37.10 way off, because we are looking at a thin slice through the chromosome.
00:24:41.02 But the good thing about this slice is
00:24:43.10 it shows us this specialization which is the kinetochore
00:24:46.05 on each of the two chromatids and it shows us
00:24:49.16 microtubules in two kinds of interactions with the chromosome.
00:24:53.05 One it is going grazing right by the kinetochore,
00:24:56.20 and in the other it is making a sort of butt end connection.
00:25:00.03 Both of these kinds of connections turn out to be important
00:25:03.10 for the process of attachment of chromosomes to the mitotic spindle.
00:25:07.16 This process has been studied experimentally in the lab of Conly Rieder.
00:25:12.19 And he set up a wonderful experimental system
00:25:15.22 where he was working with these newt cells
00:25:17.29 which we saw in the movie of the first lecture,
00:25:20.22 and they have very big chromosomes as you are seeing here.
00:25:24.13 And Rieder injected this cell with fluorescent tubulin, and you can see a single microtubule
00:25:30.20 growing here, and as that microtubule continues to grow,
00:25:34.27 because these are two frames in different times,
00:25:37.26 it actually makes contact with the chromosome shown here
00:25:42.00 as a dark ghost because there is soluble tubulin around in the background.
00:25:45.10 And this is a graph showing chromosome movement
00:25:48.15 which is diddling around without much happening
00:25:51.20 until the microtubule makes contact, and then off it goes.
00:25:55.25 So this is direct evidence for the importance of microtubule contact
00:26:00.29 with a chromosome being essential for the initial motions of the chromosome.
00:26:06.26 They've gone ahead and done electron microscopy
00:26:09.11 on one of these chromosomes that has just made contact,
00:26:12.06 and as you can see here the microtubule, diagrammed as this line,
00:26:17.10 is passing right by the kinetochore.
00:26:19.11 This appears to be one of these grazing contacts that I showed you
00:26:22.21 in that first electron micrograph of a chromosome and a spindle.
00:26:25.25 And what's going on apparently is that there are mechanical contacts
00:26:30.11 that allow the kinetochore now to motor over the surface of the microtubule
00:26:35.04 and it goes towards the pole.
00:26:36.28 The pole is where the minus end of the microtubule
00:26:40.02 resides, so this is a minus end directed movement.
00:26:43.20 Dynein has that directionality of motility and many people believe
00:26:49.02 that this interaction that we find early on in chromosomes attachment is
00:26:53.18 dynein mediated at least in some cells.
00:26:56.04 Dynein is indeed found in the spindle, and is localized at the kinetochores,
00:27:02.23 either on an isolated chromosome as shown here,
00:27:05.04 or on chromosomes as they go into mitosis in pro-metaphase.
00:27:10.14 Now this kind of evidence from antibody localization is very suggestive.
00:27:16.21 And since dynein is a minus end directed motor, you could imagine
00:27:20.23 that the dynein which is here and here is going to be
00:27:23.23 involved in pulling these chromosomes apart,
00:27:27.00 and it could be a part of the very important machinery for chromosome segregation.
00:27:35.21 The question that I want to get at, though,
00:27:37.02 before talking about the mechanics of chromosome segregation
00:27:39.26 is how do chromosomes form stable attachments to the spindle.
00:27:44.18 And the reason I focus on this, is if you look at this first sentence
00:27:48.26 which I've outlined here in red, this is a really critical point,
00:27:55.02 because the central problem of mitosis is attaching sister kinetochores to sister poles.
00:28:00.01 Once you have achieved that, all you have to do is pull the chromosomes apart
00:28:05.00 And so, how does a kinetochore know how to grab
00:28:10.23 a hold of microtubules that are coming all from
00:28:13.09 one pole or coming all from another pole
00:28:15.13 and setting up so that the pair of kinetochores is interacting with a pair of poles?
00:28:22.10 Now microtubules come at the chromosomes from both poles,
00:28:28.07 and so how does the kinetochore know which ones it should bind too?
00:28:34.14 Experiments suggest that kinetochores will bind any microtubule,
00:28:39.15 either its wall or its plus end, and they have a fairly high affinity for the plus end.
00:28:45.18 And they can't choose between east pole or west pole,
00:28:49.03 but what makes the decision is that the attachment
00:28:54.00 is going to form and become stable only when the kinetochore
00:28:57.23 microtubule junction is under tension,
00:29:00.04 that is when this kinetochore is being pulled one way,
00:29:03.06 and its sister is being pulled in the other way.
00:29:07.00 And any other form of attachment is not stable.
00:29:09.16 The evidence for this comes from some beautiful experimental work by Bruce Nicklas
00:29:14.19 whom I mentioned before as one of the master micromanipulators.
00:29:18.17 This is a grasshopper spermatocyte.
00:29:20.22 Chromosomes are shown here, and this is meiosis I
00:29:24.17 so each of these is actually a bivalent chromosome,
00:29:27.16 which makes them large and easy to work with
00:29:29.25 and there is a kinetochore over down here and a kinetochore up here.
00:29:33.16 These chromosomes are big enough and these cells are tough enough
00:29:37.04 that Nicklas has been able to manipulate them
00:29:39.18 by taking a microneedle and reaching into the cell
00:29:42.28 and interacting with the chromosome.
00:29:44.28 And I am going to show you a movie that displays this now.
00:29:47.23 There is the needle. it is coming in and interacting with this chromosome.
00:29:51.01 And the chromosome then of course starts to try to reorient,
00:29:54.01 but Nicklas comes back in and knocks it back.
00:29:56.18 And it tries to get up there again, and now he is pulling hard on that chromosome,
00:30:00.21 and pulls off that attachment, so now this chromosome is sitting there
00:30:05.04 with no spindle attachment.
00:30:07.12 What's it going to do? Well, it sort of uncoils because it probably has a little elasticity to it,
00:30:13.21 and by chance it is the other kinetochore that interacts with the spindle and now is drawn back
00:30:19.04 up towards the pole, and so what you are seeing now
00:30:22.03 is the process of congression to the metaphase plate of a bi-oriented chromosome.
00:30:28.17 in which sisters have found sister poles.
00:30:31.19 And this is known to be a stable arrangement.
00:30:35.14 How is that known, again from the work of Nicklas, now not showing movies,
00:30:40.12 but instead stills from movies.
00:30:43.01 We start here with the manipulation experiment,
00:30:45.11 and what is going on is that Bruce is going to take a chromosome
00:30:49.24 and pull it out of the spindle, and you see it down here at the bottom.
00:30:54.24 This chromosome now would re-orient just like the one we were looking at before,
00:30:59.18 but he does something different now.
00:31:00.26 He takes his microneedle and inserts it right
00:31:03.27 into that chromosome and pulls in this direction. It's diagrammed over here.
00:31:08.15 So now the chromosome is trying to make attachments with one pole down here,
00:31:14.28 and this is obviously an inappropriate attachment
00:31:18.22 because this would bring both of those two halves of the chromosome
00:31:22.00 down here to one pole, a non-disjunction.
00:31:25.10 The needle is there behaving as if it were a sister kinetochore interacting with the other pole.
00:31:31.08 And what these data show you in time is that this chromosome is stable in its mal-orientation
00:31:37.27 for a factor of ten or twenty times as long
00:31:41.11 as it would normally take a chromosomes to re-orient.
00:31:43.21 when you remove this needle, within seconds it reorients
00:31:48.01 joins the metaphase plate, and the cell goes into anaphase,
00:31:51.03 which demonstrates that his manipulation was not damaging to the cell.
00:31:55.05 So when tension is being exerted on a microtubule-chromosome interaction
00:32:01.17 it gives you a stable attachment, so what this means is that
00:32:06.18 it is only when a chromosome is a mechanical entity
00:32:10.03 is attached to opposite poles, being pulled
00:32:12.24 in opposite directions that it is under tension
00:32:15.22 and is therefore going to be subjected to these forces
00:32:19.17 that will give you the tension that gives you stability.
00:32:21.29 So accurate chromosome segregation is a selective process.
00:32:26.08 It is choosing microtubules that give tension.
00:32:30.00 It isn't organizing things so perfectly that only the
00:32:34.00 right microtubule-kinetochore connections formed.
00:32:37.18 So what generates the tension at the kinetochores?
00:32:41.09 As I've described, dynein, it could be one of the possibilities.
00:32:45.05 However, we have done antibody injection experiments
00:32:49.00 with antibodies that block dynein's motility in vitro
00:32:51.28 and they did not affect the attachment of chromosomes to the spindle.
00:32:56.20 So I don't think it is that.
00:32:59.10 Another piece of evidence is that indeed most of the dynein
00:33:02.25 leaves the kinetochore shortly after the chromosomes attach to spindle microtubules.
00:33:08.09 This means that the dynein is not their in high concentration.
00:33:12.20 in order to develop a lot of tension during much of metaphase,
00:33:16.07 and you could imagine that it isn't able to do the tension when it is not there.
00:33:21.04 On the other hand, you don't know how much dynein
00:33:23.23 you need to get the tension you are after.
00:33:26.02 So are there direct tests?
00:33:29.00 You could use dynein mutations for example,
00:33:31.05 and this is not currently possible for practical reasons, really.
00:33:35.21 Dynein is a huge protein with a very big heavy chain.
00:33:38.12 present in two copies and a large number of lighter chains
00:33:42.03 and no one has yet been able to make a temperature
00:33:46.12 sensitive mutant which would allow you to do
00:33:48.10 a temperature shift and get a quick effect.
00:33:50.20 And drug experiments always have their problems,
00:33:53.09 and so if you want to learn more about dynein
00:33:56.16 and how it is functioning, do look at the seminar by Ron Vale
00:33:59.05 which will tell you a lot about it,
00:34:01.08 but we still don't have the tools we really need
00:34:04.17 to do experiments for evaluating dynein's role in the kinetochore.
00:34:08.20 And on top of that, this diagram, which I have taken from a very nice review article
00:34:13.06 from the lab of Tim Yens shows how incredibly
00:34:16.08 complicated the biochemistry of a kinetochore is.
00:34:20.08 And we probably still have proteins that we do not yet identify,
00:34:23.26 certainly those whose function we don't understand.
00:34:27.02 So our problem is that you can imagine having a probe that interacts with dynein
00:34:32.01 or some other component here, and then there would be
00:34:34.08 indirect effects which would lead to the falling off of other essential components,
00:34:39.22 and what you'd observe is not due to what you initially perturbed,
00:34:42.25 but an indirect perturbation caused by a chain reaction.
00:34:48.05 So understanding the function of these proteins
00:34:52.04 at the kinetochore is a very hard job.
00:34:55.15 It's an important one, and indeed there are a lot of people
00:34:59.03 now working, trying to characterize all of the protein molecules that associate
00:35:03.29 with the kinetochore and trying to understand the phenotypes
00:35:07.22 of the deletion or inactivation of any one of them.
00:35:10.28 But there are always this problem of indirect effects
00:35:14.06 and it applies not only to mutants but also
00:35:16.28 to antibodies, and drug perturbation.
00:35:19.08 Many kinetochore proteins are modified during the course
00:35:23.21 of their function, by phosphorylation for example,
00:35:26.04 and what this means is that you are going to be looking at a moving target
00:35:30.28 because the function and action of the protein of interest
00:35:33.26 may change with time. So understanding the roles of all these proteins
00:35:38.11 is going to take the combined efforts of many people
00:35:40.24 using the full armamentarium of modern biology.
00:35:44.18 It's a wonderful problem and one that really deserves attention from many people.
00:35:49.03 What I want to finish up with though, now,
00:35:52.03 is a talk about one more problem,
00:35:54.22 which is the question of how do proteins get to the metaphase plate.
00:36:00.10 We've been talking about attachment, and attachment to two sister kinetochores
00:36:04.27 and if both kinetochores are being pulled to the pole you could imagine
00:36:08.13 that that creates a mechanical equilibrium,
00:36:10.27 but what pushes the chromosome to the midplane of the spindle?
00:36:14.21 And here coming back to experimental work done by Conly Rieder, we have a beautiful
00:36:19.19 example of what is certainly a part of this process
00:36:22.09 in many cells. What Rieder has done is to use a micro beam and to sever
00:36:27.19 the chromosomes at two places so the region
00:36:31.12 that has the spindle attachment point is there, and these
00:36:34.14 arms are now fragments without chromosome attachment points,
00:36:38.18 so called acentric fragments. And over the course of time,
00:36:42.13 one can see their behavior. They are pushed away from the pole
00:36:45.20 and eliminated from this monopolar situation.
00:36:49.29 What this suggests is that each pole is pushing
00:36:52.29 on all of the objects of the spindle, even as the kinetochores are being pulled towards the pole.
00:36:59.14 And it suggests that what we may have here is a situation
00:37:02.27 where chromosome mechanics is a balance between pole directed forces acting on kinetochores
00:37:09.27 and pushing forces acting on the body of the chromosome as a whole.
00:37:16.01 This is probably part of the story, and in some cells
00:37:19.15 it may be much of the story of understanding pro-metaphase.
00:37:23.04 because you can see here what we have is a pair of fibers
00:37:27.11 that are attaching to the chromosomes
00:37:29.02 and they are pulling towards the poles, and then I'm diagramming
00:37:32.26 the pushing forces that are pushing away from the poles,
00:37:36.16 and you want to know of course,
00:37:38.00 where do these pushing forces come from?
00:37:40.02 The best evidence at the moment is that they come from another
00:37:44.03 motor protein, a kind of kinesin that is often called a chromo-kinesin
00:37:48.02 because it binds to chromosomes and it interacts with microtubules
00:37:52.00 and it walks in the plus end direction. So it
00:37:55.11 may be pushing the chromosomes away from the poles, contributing to this force
00:38:00.17 that aligns the chromosomes at the metaphase plate.
00:38:04.10 Mitosis still provides lots of problems for interested biologists.
00:38:10.10 We really want to know the biochemical basis of each of the spindle functions.
00:38:14.10 The chromosome attachment, the congression to the metaphase plate,
00:38:18.00 the regulation of anaphase onset, and the mechanism of chromosome to pole motion.
00:38:23.01 Each of these is a complex cellular event involving
00:38:26.27 many, many proteins working together and it will take a consortium of people interested
00:38:32.08 in individual molecules and people interested in processes to work it out.
00:38:37.05 And what I'll talk about in the next lecture
00:38:39.04 is how our lab is approaching this kind of complexity
00:38:42.19 in order to try to understand a specific subset
00:38:46.09 of the problems: the motion of chromosomes to the poles.

00:00:10.29 Hello, I am Dick McIntosh, professor of Cell Biology at the University of Colorado
00:00:14.24 in Boulder. This is the third of three lectures that I am going to give on the subject of chromosome
00:00:20.27 movement. And in this one, I am going to build on information which you've seen
00:00:26.06 from the previous lectures in order to talk about this single
00:00:30.05 problem of how do chromosomes approach the poles
00:00:33.13 during anaphase--the process called Anaphase A.
00:00:40.00 Anaphase A is an essential part of chromosome segregation
00:00:46.20 in most cells. And there's a wide range of evidence from different experimental methods
00:00:53.03 that people have applied, and if you've seen the previous lecture
00:00:55.21 you got a little flavor of just how broad
00:00:59.11 the experimental landscape is and people have used in order to try to understand mitotic processes.
00:01:06.02 And this account that I am going to give now is
00:01:09.27 quite a personal one in that it is going to based on recent work from our lab
00:01:13.29 and it's of course, a limited perspective.
00:01:16.09 Because any individual's approach to a scientific problem is going to look at only a part of it
00:01:22.09 because that is the way you can dig deeply enough in order to try to make some progress.
00:01:26.06 But, nonetheless, I hope that what I will be able to convey to you
00:01:31.12 is the ways in which you could use different
00:01:33.22 approaches to get pretty deep and maybe come close to understanding
00:01:38.16 a fundamental biological process.
00:01:40.06 So how do chromosomes approach the poles in anaphase A?
00:01:46.12 There have been two important hypotheses that have been very active
00:01:50.17 in this field for a long time. The Motor Hypothesis in which enzymes,
00:01:54.21 for example, dynein that we were discussing last time
00:01:57.18 or a kinesin could be involved in driving the chromosomes to the poles,
00:02:02.18 as a motile process just as those same motors move vesicles around.
00:02:06.11 in cells, for example. But it's clear when you look at mitosis
00:02:10.23 that microtubules must shorten during Anaphase A.
00:02:14.21 That is what it means to approach the poles, and this has given rise
00:02:17.26 to the Depolymerization Hypothesis, which originally was formulated by Gunnar Ostergren
00:02:25.04 back in the 1940's and 50's and then pioneered by Shinya Inoue
00:02:29.19 based on some beautiful work that he did with polarizing microscopy
00:02:33.07 and I was very much a motor hypothesis man for much of my career,
00:02:37.28 because it seemed just such an attractive way of thinking about this complex motile process.
00:02:43.24 But, what I am going to show you today is that I've switched sides, and I've come to believe
00:02:49.01 that depolymerization may well be at the root of chromosome movement.
00:02:54.04 Now, how do you test a hypothesis with a complicated process like mitosis?
00:03:02.11 The obvious way would be to inactivate a motor protein so that it will make problems
00:03:10.09 for the mitotic process and you'll be able to see
00:03:13.13 what are the ways in which chromosomes move or don't move
00:03:16.12 under circumstances where they no longer have this particular motor
00:03:19.21 function. What's been seen by a number of people
00:03:22.25 who have taken this approach either genetically or pharmacologically
00:03:26.00 is that if you can get a spindle built, and you can get the chromosomes
00:03:29.17 there so that you can now study Anaphase A,
00:03:32.21 if you perturb the function of a motor, you mess up aspects of
00:03:40.01 spindle function. And this messing up can show up in several ways,
00:03:43.23 It can show up in the failure of the spindle to retain
00:03:46.23 its structural integrity
00:03:48.05 or it can show up in the fact that if you have a way of measuring how frequently a chromosome is lost,
00:03:55.25 then chromosomes are lost more frequently when a motor
00:03:57.14 is missing. The remarkable thing though is when you look at a lot of the data that is in the literature
00:04:02.25 many aspects of mitosis continue even when a given motor is perturbed.
00:04:09.00 They go a little slower, not all the chromosomes may segregate
00:04:13.04 properly, but many of them do.
00:04:15.19 And of course if you are interested in the importance of your own work,
00:04:19.10 what you want to do if you've made a perturbation is see it as causing a lot of trouble.
00:04:23.18 And so you emphasize the things that are not working.
00:04:25.28 But what I've been doing in my own mind, and in the work we are doing in our lab
00:04:30.14 is asking what is still going on even when motors are not there.
00:04:35.18 And the way in which we've done this is to turn to yeast cells
00:04:38.17 where it is possible to do gene deletions comparatively easily.
00:04:43.00 and prove to yourself that the entire piece of DNA is gone from
00:04:47.14 the cell. So there is no possibility that the motor which is the protein
00:04:51.07 product of that gene is contributing to the mitotic process.
00:04:54.08 that you are seeing. And then you can ask: What happens?
00:04:58.02 And we've done this in a fission yeast cell using
00:05:02.00 the typical molecular techniques to delete
00:05:05.20 Two kinesin like proteins, each of which is a minus end in its directed activity
00:05:11.26 and the dynein heavy chain, and so there are no minus end directed
00:05:16.27 motors left in this cell, and how do we know that?
00:05:20.01 Well, the genome of this organism is sequenced
00:05:22.03 and we know that, as well as we understand, motor function with microtubules, there is no motor left
00:05:28.16 in the cell, and yet, when I know show you
00:05:32.04 the motion of the poles and one chromosome,
00:05:35.10 which we've marked with a fluorescent tag near the kinetochore,
00:05:39.15 this time lapse will show the separation of the spindle poles,
00:05:43.21 and you can see the chromosome, which is here, and you are going
00:05:46.11 to see it migrate towards the spindle pole
00:05:49.11 and we can measure the speed at which it migrates towards the pole,
00:05:52.20 as it becomes attached to the spindle.
00:05:55.05 Now of course, it should bi-orient and come to the metaphase plate,
00:05:59.07 this particular chromosome was a little tardy in this,
00:06:02.27 and that's common with these chromosomes that lack several motors, but you could
00:06:06.26 see it did actually segregate correctly
00:06:09.20 and from this kind of raw data we've been able to
00:06:13.00 measure the speed of the final approach of a chromosome
00:06:17.05 to the pole in a variety of genotypes.
00:06:20.05 Wildtype shown in yellow
00:06:21.18 and then one motor mutant after another and down here
00:06:26.04 in green we are seeing a deletion of all three minus end directed motors
00:06:32.05 and yet this final approach to the pole
00:06:34.27 is occurring at a speed which is no different from wildtype.
00:06:38.13 So these cells make mistakes. They are not healthy, and they would not
00:06:42.21 survive in the wild, and indeed we've measured the frequency of chromosome loss
00:06:46.27 and it's up by several, well even hundreds of fold.
00:06:52.12 So this is not a healthy organism
00:06:54.05 but it's an organism in which chromosome to pole motion occurs at a speed which is
00:06:59.20 undistinguishable from the normal wildtype chromosome movement.
00:07:04.20 That means these motors are not important for such motion.
00:07:08.12 They may be important for other things, like attaching
00:07:10.20 the chromosomes to the spindle, or
00:07:12.22 segregation or integrity of the spindle
00:07:15.01 poles, but not for this fundamental phenomenon of anaphase A.
00:07:19.27 So what...well, first of all we can say this is not simply due to fission yeast
00:07:25.20 idiosyncracies, it is also true in budding yeast,
00:07:28.23 where the Tanaka group has been able to demonstrate
00:07:31.11 this quite clearly. So, these motions must be caused
00:07:35.02 by some thing that is going on in this cell
00:07:38.08 which is not a minus end directed motor.
00:07:41.06 it could be some non microtubule component
00:07:43.21 of the spindle, but this doesn't seem very likely
00:07:45.24 because although there have been the identification of
00:07:49.21 actin in the spindle, it turns out that the actin is generally not
00:07:52.22 fibrous when it is in this spindle.
00:07:53.28 And there's been the identification of matrices in the spindle,
00:07:58.06 but these matrices are not yet known to have any kinetic function.
00:08:01.17 And it could be then that it is simply microtubule depolymerization,
00:08:05.20 which is itself a motor in some way.
00:08:08.19 How do you find out?
00:08:11.17 This is an implausible idea, and in order to convince anybody
00:08:16.14 we really need some strong experimental evidence.
00:08:19.03 The implausibility of this was described best to me
00:08:22.27 by the expert kineticist who studied myosin and other enzymes, Ed Taylor,
00:08:28.15 who was very skeptical about the disassembly hypothesis, and he
00:08:32.09 pointed out that if you were a rock climber suspended on a rope
00:08:36.08 from a cliff and you wanted to go up the cliff, you certainly wouldn't do it by
00:08:40.24 lopping off the rope to make it shorter.
00:08:42.19 And this analogy certainly casts doubt on the hypothesis.
00:08:47.28 But what I am going to show you is that it has some validity to it.
00:08:52.16 Microtubule polymerization can do mechanical work.
00:08:56.12 And this was shown very nicely in the laboratory of Hotani, many years ago
00:09:01.04 where they put soluble tubulin inside lipid vesicles
00:09:04.26 and induced it to polymerize
00:09:06.19 and the polymerization of tubulin drove these deformations of the lipid membrane
00:09:11.21 showing that polymerization could do work.
00:09:14.04 And indeed it is now well known that actin polymerization can do work
00:09:18.21 and you should look at the iBio seminar that deals with this very nicely
00:09:24.21 because it has a beautiful amount of detail, all shown by Julie Theriot.
00:09:30.23 So polymerization is easy to understand
00:09:34.22 as a motor, but what about depolymerization?
00:09:38.08 We designed an experimental system in which to look at this
00:09:41.17 in which we had an objective lense on the microscope, and a coverslip
00:09:44.25 we were looking at an object which was sort of our
00:09:48.21 in vitro manifestation of a spindle pole,
00:09:50.23 we happened to use a ciliated protozoan
00:09:54.20 that we lysed with a detergent to wash away the membrane and clean out the cytoplasm
00:09:59.05 but it left behind what is called a pellicle
00:10:01.22 which has about 500 basal bodies for flagella
00:10:05.24 and that structure will now nucleate large numbers of microtubules.
00:10:10.05 We used purified brain tubulin to flow it in and get this forest of microtubules
00:10:15.11 all of whose plus ends are pointing away
00:10:18.09 from the organizer, just as the spindle microtubule plus ends
00:10:21.22 are pointing away from the centrosome.
00:10:24.07 We could then flow in chromosomes that we had
00:10:27.13 partially purified from CHO cells and ask, "Do they bind?
00:10:33.11 And if they bind, can we make them move?"
00:10:34.17 And in this movie taken by Vivian Lombillo taken when she was a graduate student
00:10:39.09 in the lab, you can see a pair of chromosomes that are caught in the microtubule
00:10:45.05 forest that has grown from this pellicle,
00:10:47.22 and as I run the movie, we will now flow in buffer
00:10:51.02 which contains no tubulin, and you can see the chromosome immediately wash down stream
00:10:56.20 in the flow of the buffer. This movie is real time, so
00:11:00.09 we are not exaggerating any speeds and the force of the flow
00:11:03.29 is substantial, and yet, when they come into focus,
00:11:06.18 you can see that the chromsomes are still attached.
00:11:09.08 This buffer contains no nucleotide triphosphate, no ATP, no GTP
00:11:14.20 and in fact it contains a pyrase, an enzyme that drops the concentration of nucleotide below nanomolar
00:11:22.11 and yet these chromosomes move into this structure here as the microtubules disassemble
00:11:28.29 at speeds which are even on the high side for physiological motion of chromosomes.
00:11:34.19 So this work demonstrates that microtubule depolymerization
00:11:39.06 without any ATP dependent motor activity
00:11:42.04 can move chromosomes in a test tube.
00:11:45.19 So we've seen that chromosomes can approach the poles
00:11:49.01 in vivo with no motors present in the cell
00:11:52.17 and they can approach this spindle pole here
00:11:55.22 when we have no motors present and fed a fuel that could move them.
00:12:02.05 There may of course still be motor enzymes.
00:12:04.09 on the kinetochores, but this cannot be an ATP dependent motor activity.
00:12:08.09 so we interpret this as a disassembly dependent motility.
00:12:13.13 How could depolymerization of a fiber cause movement?
00:12:17.24 These images which are electron micrographs taken
00:12:21.19 of frozen hydrated microtubules by Eva and Eckhart Mandelkow
00:12:27.14 collaborating with Ron Milligan show what the ends of polymerizing microtubules
00:12:32.10 look like. They are quite blunt.
00:12:35.01 On the other hand depolymerizing microtubules show
00:12:38.10 this characteristic curl to the tip of the microtubule
00:12:42.09 where a strand of tubulin, a protofilament, is bending.
00:12:47.11 And this appears to be a characteristic event of the disassembly process.
00:12:52.14 Where does this come from?
00:12:54.07 Well it is related to the cycle of polymerization and depolymerization of tubulin.
00:12:59.26 Tubulin to polymerize has GTP bound and
00:13:04.00 the molecule is more or less straight and it adds onto the ends of the microtubule.
00:13:08.07 But the microtubule activates the GTPase activity of tubulin,
00:13:13.02 it is like a GTPase activating factor when you think about G proteins.
00:13:17.01 And so the majority of the tubulin in the microtubule is GDP tubulin.
00:13:23.02 And that irony is that GDP tubulin will not polymerize.
00:13:27.25 The GDP tubulin tends to fall apart.
00:13:31.06 and indeed, as it falls apart it shows this curvature
00:13:34.20 and the interpretation that has been put on this by a
00:13:37.07 number of investigators working on it
00:13:39.13 principally Eva Nogales, is that the tubulin molecule in the GTP bound state
00:13:45.27 tends to be more or less straight, but in the GDP bound state
00:13:49.18 it tends to bend, and this bending means that when GDP is in the wall of the microtuble
00:13:56.08 it's under strain as a result of interactions with neighboring tubulin molecules
00:14:01.23 that interact with it by non-covalent bonds.
00:14:05.21 So those interactions are keeping the molecule constrained and straight,
00:14:10.03 unless you are at an end without any GTP tubulin
00:14:13.13 on the end to provide straightness.
00:14:16.02 And now the curvature of these tubulin protofilaments is a relaxation of
00:14:21.21 tubulin GDP molecule to its minimum energy geometry.
00:14:27.02 What this means is that a microtubule in the course of depolymerization
00:14:33.17 is going to have a wave of conformational change.
00:14:38.11 Doug Koshland was the first person to point out that this conformational wave
00:14:43.27 might be a way of pushing on things
00:14:47.00 and it might help to pull chromosomes to the pole.
00:14:49.11 But of course, the cell to take advantage of it must find some way
00:14:53.17 to couple to this microtubule so it can grasp the microtubule
00:14:58.00 and experience the force from those bending protofilaments.
00:15:01.13 We need to understand what that coupler might be in order to see how this relaxation
00:15:07.23 of the tubulin molecule could be a power stroke that would drive chromosome movement.
00:15:12.03 The first indication as to what this might be
00:15:16.03 came from some more work done by Vivian with that experimental system that I showed you earlier
00:15:20.21 with the chromosomes moving in vitro.
00:15:23.09 She added antibodies to kinesin, first a general kinesin,
00:15:28.09 and then a kinesin that is specifically localized at kinetochores
00:15:31.17 so called centromere protein E.
00:15:33.28 And those antibodies caused a dramatic reduction
00:15:37.25 in the motion of the chromosomes in this depolymerization
00:15:42.08 dependent fashion, suggesting that a kinetochore motor
00:15:46.28 is important for depolymerization dependent movement even when no ATP is present.
00:15:52.26 But remember the caveat that I raised at the end of the last lecture
00:15:57.25 that even when you add antibodies that are monospecific
00:16:01.19 they may have indirect effects. So this doesn't really prove to us that this molecule
00:16:06.24 is a coupler or that it is working in this way.
00:16:09.24 It is strong suggestive evidence.
00:16:11.19 We've obtained other evidence, however,
00:16:14.06 that a microtubule dependent motor enzyme can
00:16:18.06 work as a coupler using a kinesin-8 from pombe cells.
00:16:23.03 And if that kinesin is attached to a bead and the bead is then allowed to interact with a microtubule
00:16:32.05 which isn't visible here, but it is shown in diagrammatic form over here
00:16:35.25 and we now induce the microtubules
00:16:37.26 to disassemble, the bead is pulled by microtubule disassembly
00:16:42.01 just as I have shown you in those other experiments
00:16:44.29 and these graphs show the rate and the trajectory.
00:16:48.05 Clearly this is quite a processive movement.
00:16:50.26 In the sense that the bead is following the disassembly microtubule end for quite a distance.
00:16:56.03 So a motor enzyme can serve as an ATP independent coupling factor
00:17:01.29 to bind a cargo to a disassembling microtubule.
00:17:05.25 Does it have to be a motor? No.
00:17:09.02 And one of the most remarkable discoveries in the mitosis field
00:17:12.09 recently has been the discovery of this complex called either Dam1 or DASH
00:17:19.23 depending on whose laboratory you happen to have been associated with.
00:17:23.24 The people who first discovered and named the Dam1 protein
00:17:27.04 and then gradually found more and more proteins that were part of a big protein complex
00:17:31.26 call it the Dam1 complex. 'veI collaborated with that lab,
00:17:35.19 so I'll use that name, but the name DASH is used
00:17:38.29 by many other labs for the same complex.
00:17:40.29 It's an unusual complex because it involves
00:17:44.21 ten different polypeptides all of which
00:17:47.17 assemble into a little football shaped object
00:17:50.13 and this is called the Dam1 complex.
00:17:52.24 And this complex polymerizes with others of its own kind
00:17:56.27 and those polymers form rings around microtubules.
00:18:00.22 Here are the rings forming just on the surface of a support
00:18:04.17 visualized in the electron microscope just by negative staining.
00:18:07.06 And some really excellent work both by the Westermann group where Nogales
00:18:13.20 has been doing the electron microscopy
00:18:15.15 in her lab and in the group that's at Cambridge, Massachussetts
00:18:20.07 under Steve Harrison has been...they've been providing
00:18:23.19 expert and excellent evidence about
00:18:26.20 the structure of this complex and the way in which
00:18:29.06 it interacts with microtubules.
00:18:31.02 We've collaborated with the Berkeley group
00:18:34.09 which included Westermann and his mentors, Georjana Barnes and David Drubin
00:18:40.00 and with them we've also been able to purify this complex and
00:18:43.28 label it with fluorescent dye, and allow it to interact with microtubules
00:18:49.13 in our in vitro system, where this is that pellicle
00:18:52.01 that you've been seeing
00:18:53.02 and these then are complexes of the Dam1 protein,
00:18:56.09 which are fluorescent. This is
00:18:58.18 what our Dam1 complex looks like when it surrounds the microtubules seen
00:19:02.02 in the electron microscope, but of course you can't do kinetic experiments
00:19:05.19 in the electron microscope. So we are going to do an experiment here
00:19:09.17 which we watch what happens to these Dam1 complexes
00:19:13.01 when we cause the microtubules to disassemble.
00:19:15.19 And here we will now bleach the little tip
00:19:18.19 that's on the end of the microtubule, and the microtubule
00:19:20.25 starts to depolymerize, and you can see that the Dam1 complex
00:19:25.05 is moved with the ends of the microtubule as the ends of the microtubule
00:19:29.09 shortens. So the Dam1 complex
00:19:32.14 is also a coupler that can take advantage of the structures
00:19:38.02 that are found at the end of the microtubule.
00:19:40.17 This coupler can actually pull a load, and
00:19:45.21 what we've done here is to put the Dam1 complex
00:19:48.16 onto a bead. And this bead now can be followed
00:19:54.05 as an object which is a load for the Dam1 complex
00:19:57.12 to move as it associates with the microtubule.
00:20:00.25 And again, when we induce microtubule disassembly, as the disassembly
00:20:04.23 reaches the bead, the bead will stop its Brownian movement
00:20:09.04 just back and forth and will start a progressive motion
00:20:12.18 towards the origin of the microtubules, which is that pellicle sitting over at the side.
00:20:17.24 Now this kind of work allowed us to determine that the Dam1 complex seems to
00:20:24.18 form a variety of structures, all of which can interact with the microtubules.
00:20:28.23 If we attach Dam1 to a bead, and do not have the Dam1 complex in solution,
00:20:36.00 but instead just allow the bead to bind to the microtubule,
00:20:39.27 we get a distribution of velocities that is shown here in red.
00:20:43.07 If we have Dam1 in solution, so that a complex can form that would make this ring
00:20:50.10 shaped structures that I showed you in the electron microscope,
00:20:52.29 what we see is that we still get bead movement, but the bead movement is slower,
00:20:57.15 as if the formation of that ring might actually retard the rate of disassembly of the microtubules.
00:21:05.06 So this looks likea process that needs detailed study,
00:21:08.14 and we've done enough experiments that I won't be able to describe them all to you.
00:21:11.16 by any means in the course of this short lecture,
00:21:14.07 but I want to show you the tool that has been the most important to us in
00:21:17.25 trying to do this kind of work. It is a standard light microscope, which has
00:21:22.29 a sensitive camera at its top.
00:21:25.11 and then over here it has a couple of lasers, one of which
00:21:29.06 is very strong and can be led through a device that allows you
00:21:33.11 to steer the laser beam and then up into the microscope
00:21:36.14 the other laser that is over there is just to help us align things.
00:21:39.26 So a laser beam is coming down through our objective lense
00:21:43.07 and this very bright light can be used in what is called an optical trap.
00:21:47.02 A device where you can grab a small object
00:21:50.19 that refracts light, like a bead of glass or plastic.
00:21:54.00 Over on this side, we have two other lasers, one green and one blue,
00:21:58.22 that we use for bleaching the fluorescence
00:22:01.20 of some parts of our specimen. And what
00:22:04.11 we are going to use is tricks in order to
00:22:06.19 be able to do experiments on beads and ask:
00:22:11.00 Can we monitor the force that is generated in this system?
00:22:13.18 So here's again our pellicle serving as a nucleator;
00:22:17.13 microtubules growing from purified tubulin.
00:22:19.16 The problem is that these microtubules have to be labile.
00:22:22.27 And that means that if we dilute the preparation of
00:22:26.21 tubulin by washing anything else in
00:22:29.08 we are going to cause them to disassemble, so we need to stabilize them.
00:22:32.25 And we do that by putting a cap on the
00:22:35.08 tip of them where we polymerize the tubulin
00:22:38.02 in an analog of GTP which does not hydrolyse, or hydrolyzes very slowly.
00:22:42.05 And the result is that we have now stable microtubules
00:22:46.04 so we can drop the tubulin concentration to zero and
00:22:49.26 now we can bring in beads coated with
00:22:52.26 something that will make them stick to the microtubule itself.
00:22:55.28 The way we do this is we use biotinylated tubulin
00:22:59.01 and beads coated with the protein avidin, so the
00:23:01.27 connection between the microtubule and the bead
00:23:05.02 is one of the strongest non-covalent interactions known in biology
00:23:08.25 and this is not going anywhere, it's certainly not
00:23:11.25 a motor and we can ask: when the microtubule disassembles,
00:23:15.25 what happens? And the way in which we do this experiment is
00:23:19.02 to turn on our laser trap so that this bright laser light is holding that
00:23:24.14 bead and we have a way of measuring the position of the center of the bead
00:23:29.04 very accurately and then we turn on our photobleaching laser
00:23:32.15 in order to inactivate the tubulin that is there, and the cap comes off, and the microtubule
00:23:38.27 will disassemble, and you may have noticed that I drew in just a little
00:23:42.13 bit of a movement there of that bead
00:23:44.17 as the disassembly went by.
00:23:46.24 That's the kind of event we are looking for
00:23:48.11 in order to take motors completely out of the equation
00:23:51.26 and ask: can microtubule disassembly do work?
00:23:55.02 And the answer is yes.
00:23:57.13 Here is a cartoon of what we think is going on
00:24:00.14 with the bead drawn very small relative to the microtubule,
00:24:03.28 and you can see the bending protofilament that could be
00:24:06.08 exerting a force on the bead.
00:24:09.04 This is a trace that we get from a very sensitive device
00:24:11.24 called a quadrant photo detector which is allowing us
00:24:14.22 to determine the position of the center of the
00:24:16.26 bead extremely accurately to within a nanometer or so.
00:24:20.15 And all of this wiggling you see
00:24:22.04 here is the thermal noise that the bead
00:24:24.23 is oscillating as a result of interactions with molecules
00:24:28.05 in solution. But as the disassembly occurs,
00:24:31.00 the bead is pushed a little bit toward the minus ends of the microtubule, and then released.
00:24:35.09 and now it is simply in the center of the trap, and the microtubule has
00:24:39.14 disappeared. Now if this really is
00:24:43.04 a force being generated in this way, you could imagine that we are pulling
00:24:48.15 here on the center of the bead, with our trap, and the radius of the bead then
00:24:53.00 is like a lever arm. And the smaller the bead, the less our mechanical advantage
00:24:58.18 and the bigger the force that we should generate, and indeed
00:25:01.25 here you can see with a 2 micron bead
00:25:03.25 versus a 1 micron bead versus a half micron bead
00:25:06.17 we get bigger forces as the bead gets smaller,
00:25:09.25 suggesting that this really is the bending of the protofilament
00:25:13.09 pushing on the bead to give us the force that we are seeing.
00:25:16.07 How much force? Not very much.
00:25:18.27 with this non-physiological system. But, we also presume that we are only interacting
00:25:24.27 with one side of a microtubule, and if you think
00:25:28.04 about the Dam1 complex, which surrounds the microtubule
00:25:31.16 as a ring, one could imagine that it would experience force from all the bending protofilaments
00:25:36.10 at once, and would give you significantly more force.
00:25:39.24 So we have naturally gone ahead and tried to attach the beads
00:25:43.07 to Dam1 complex where here this is a real micrograph of
00:25:47.22 the Dam1 complex on a microtubule, but this is just a cartoon
00:25:51.11 with representations of the antibodies that we have bound to the bead.
00:25:56.24 And we are using antibodies that interact with the Dam1 complex,
00:26:00.21 and give us quite a tight bond, and now we can ask, "When disassembly comes by, what do we see?"
00:26:06.08 And the answer is a longer and much stronger force.
00:26:09.24 This force is now about six times as great as the force that we observed
00:26:14.07 when we were only sampling one side of the microtubule.
00:26:17.11 And so it really looks as if a ring
00:26:19.28 is surrounding the microtubule and
00:26:22.19 sampling the action of all the protofilaments
00:26:25.04 and producing a force, which, once we've made corrections
00:26:28.00 for the bead diameter, looks as if it would be 20, 30, even 40 piconewtons,
00:26:33.18 which is an unusual unit of force for most of you,
00:26:36.23 but a kinesin molecule or a dynein molecule develops somewhere around 5 piconewtons.
00:26:42.04 So a microtubule interacting with a ring
00:26:45.13 is really powerful. It is sort of like a bulldozer
00:26:48.20 and it's no wonder that you can delete motors from the cell
00:26:51.17 and chromosomes will continue to move, if this is the process that
00:26:55.04 is really doing it. So how do these things work?
00:26:58.28 Well, here are the two hypotheses that are central to the way people are thinking about this movement
00:27:04.09 at this point. On the left you are seeing
00:27:06.23 a Brownian movement, a random walk by diffusion of a ring which is modeled here in these accurate simulations
00:27:15.15 done by my colleague, Fazly Ataullakhanov and his students in Moscow
00:27:20.02 and they are allowing the diffusion that would occur with an object of
00:27:25.28 the size of the ring and loose binding
00:27:27.14 and this diffusion still can give rise to processive movement
00:27:32.16 because the diffusion is biased
00:27:34.22 by the disassembly of the microtubule.
00:27:37.05 Over here we are showing a different model
00:27:40.05 And this is one in which the ring is presumed to bind
00:27:43.17 tightly to the wall of the microtubule, noncovalently,
00:27:47.03 but still tightly, so that as these protofilaments
00:27:49.29 bend they have to force the ring from one position to the next position.
00:27:54.04 to the next, and you can see kind of stall as it is going
00:27:57.10 and then it will go ahead and go farther again.
00:27:59.24 This is then a situation where a forced walk
00:28:04.00 is causing the migration of the ring.
00:28:06.19 Now intuitively you might think that this biased diffusion is a more efficient
00:28:11.15 system because you are not having to expend so much force in overcoming the binding
00:28:16.02 energy between the ring and the microtubule.
00:28:17.27 But this has problems, because polymerization and depolymerization
00:28:23.29 of microtubules are molecular events which occur
00:28:26.17 with random fluctuations, and so every now and then
00:28:29.24 the depolymerization pauses and the ends of the microtubule presumably
00:28:35.10 go straight for a little while, and if there were a load on this ring, it might just pull right off
00:28:40.10 the end of the microtubule which would be a terrible catastrophe
00:28:44.13 if you were trying to move chromosomes by microtubule disassembly.
00:28:49.23 So intuitively we favor this tight binding model
00:28:53.23 but indeed there is quite a bit of evidence that
00:28:56.12 that is the case.
00:28:58.25 So is the Dam1 ring then the answer
00:29:01.24 for how you couple chromosomes to microtubules?
00:29:04.11 Umm, in budding yeast, Dam1 is in the spindle, it binds to chromatin.
00:29:11.04 It is essential for proper chromosome segregation
00:29:13.14 and this says that the Dam1 complex is an excellent candidate
00:29:18.23 for the coupler in budding yeast.
00:29:22.05 Interestingly, in fission yeast, it is no longer essential
00:29:27.03 in cells that are otherwise wildtype.
00:29:29.12 Now if you start deleting mitotic motors
00:29:32.01 then the Dam1 complex becomes essential
00:29:34.29 but what we have here is a situation where in just going from
00:29:37.27 one ascomycete fungus to another,
00:29:41.11 we've moved from essential to contributory.
00:29:44.19 Another difference between these two spindles
00:29:48.01 is that in budding yeast there is one microtubule per kinetochore
00:29:52.06 whereas in fission yeast there are 2 to 4 microtubules
00:29:55.23 per kinetochore, so maybe, the Dam1 complex
00:29:58.27 is absolutely essential when you need to regulate the disassembly of microtubules
00:30:04.02 so tightly that you don't let the microtubule end get away from the kinetochore
00:30:08.10 but when you have more microtubules
00:30:11.07 and you have others that you can rely on, it is no longer an essential process.
00:30:15.11 Even more disturbing in terms of thinking about the generality of the Dam1 complex
00:30:20.18 is that outside the fungi, this protein has not yet been found.
00:30:23.24 Now this doesn't say that other rings are not going to be found, and a number of
00:30:28.12 possibilities have been detected becasue the Dam1 complex is so appealing
00:30:33.07 as a way of doing this job well that many scientists feel that rings must be the answer
00:30:38.23 and they are trying to find the components of the rings
00:30:41.26 in other cell types.
00:30:43.12 We've taken a different approach, which is
00:30:45.09 to go and look at the kinetochore-microtubule connection
00:30:48.19 with the best structural tools
00:30:50.11 that are available, and ask, "What do we find?"
00:30:52.24 And the way we've done this is to use
00:30:55.04 electron tomography. Now this is an electron micrograph
00:30:58.26 of a chromosome and a spindle fiber, although it is very unprepossessing
00:31:03.07 and the reason for it is that it is one of series of
00:31:06.01 images from a tilt series where we now have a thick sample
00:31:10.20 about three or four hundred nanometers thick
00:31:12.26 in the electron microscope tipping back and forth
00:31:15.27 and we are collecting images at 1 degree intervals from +70 to -70 degrees
00:31:21.17 and then we go ahead and tip around the orthogonal axis
00:31:25.01 in order to collect a large number of
00:31:27.09 views of this three dimensional object.
00:31:30.15 These can then be combined by a variety of mathematical approaches
00:31:34.13 to create what is called a tomogram, or a 3 dimensional reconstruction of all the material
00:31:40.10 that was in that region that we were imaging.
00:31:43.15 Chromatin down here. Kinetochore here.
00:31:45.20 Microtubules there and you can see flared ends
00:31:49.03 on the tips of many of the microtubules in this array.
00:31:52.15 We can then use software to pull out a single
00:31:55.29 slice from our three dimensional reconstruction
00:31:58.03 that contains the axes of one or more
00:32:00.24 microtubules, so that we can see just exactly what these ends look like.
00:32:05.25 These ends are in some way attached to the chromosome,
00:32:11.01 and what we want to know is how.
00:32:14.00 And by just taking a descriptive approach,
00:32:16.23 we can try to get insight, and tomography allows a completly novel way of looking at this
00:32:22.12 and what I am going to show you now is a series
00:32:24.23 of images in which I took a plane
00:32:26.27 that contained the microtubule axis and then
00:32:29.24 I am going to rotate that plane around the microtubule
00:32:32.16 axis, so that we can visualize a single microtubule from multiple orientaitons.
00:32:38.23 And you can see flaring protofilaments come and go.
00:32:42.02 There is one waving way up here, here is one coming down.
00:32:46.01 All of these are images at different orientations.
00:32:47.29 We can then extract this structural information
00:32:51.27 as a series of graphics, which allow us, then, to see
00:32:55.22 a representation of the protofilaments and their
00:32:58.25 flare at the kinetochore end of one kinetochore microtubule.
00:33:03.29 This has allowed us both to quantify
00:33:06.28 exactly the shape of these flares to within
00:33:09.29 the precision of our methods for preserving
00:33:12.17 this sample, and also to ask, "What is connected
00:33:16.05 to those flares?" Well, in the first instance, when we
00:33:20.15 look at the flares of the protofilaments from kinetochore microtubules
00:33:24.17 here, and non kinetochore microtubules here
00:33:28.15 and then compare them with depolymerizing and polymerizing
00:33:32.01 microtubules in vitro, imaged in that study that I showed you before
00:33:37.14 by the Mandelkows and Ron Milligan.
00:33:40.02 What you can see is that there is a tremendous range in the structure of the protofilament
00:33:44.21 of both kinetochore and non-kinetochore microtubules
00:33:48.00 They are sort of in between assembling and disassembling.
00:33:52.09 And this was quite hard to understand; however, what we've
00:33:56.00 done is to focus in on the bending protofilaments which are
00:34:02.27 in an intermediate group, and what we can find
00:34:05.23 is that many of them have little strands that connect from
00:34:09.29 the protofilament itself up into the region of the chromatin.
00:34:13.10 And then in the lower half of this slide, what I've done is to
00:34:16.06 put graphic objects down to show you
00:34:19.29 what I think are both the protofilaments themselves
00:34:22.22 and these little fibrils that are connected to the protofilamants and also connected
00:34:28.06 up into the chromatin. And we are calling these little fibrils
00:34:31.19 kinetochore fibrils, for obvious reasons.
00:34:34.21 The trouble is that this imaging is right at the limit
00:34:39.16 of the methodology, both are ability to preserve the sample
00:34:42.17 well because we are looking at a whole cell here, which has been prepared for electron microscopy
00:34:47.07 and the imaging resolution of the methods
00:34:50.02 that we are using-tomography of these thick samples.
00:34:53.19 It would be very nice if we could average things up in order to try to see
00:34:58.04 whether there is an averaged structure for the fibril.
00:35:00.25 And Katya Grishchuk in the lab had the insight that if we were able to
00:35:06.05 take the intermediate classes of protofilaments,
00:35:10.16 yes, in between polymerizing and depolymerizing
00:35:14.08 and look at these only, we might see something special
00:35:18.13 because these are not simply depolymerizing
00:35:21.06 and so we took those, sorted objectively, simply on the basis of their slope near the microtubule
00:35:27.12 wall. We could then take forty or fifty
00:35:30.19 such protofilaments from the original image data and average them all.
00:35:36.00 And this is such an average for a non-kinetochore microtubule.
00:35:38.22 This is a metaphase kinetochore microtubule
00:35:41.18 from that intermediate group, and I think you can
00:35:43.14 see now there is a very respectable fiber that is averaged
00:35:47.14 up out of these fifty or so data sets.
00:35:49.28 This is also found in anaphase, where the flare is slightly longer.
00:35:53.29 on this protofilament, whereas the ram's horn groups-the ones that have the big curvature
00:36:00.17 characteristic of depolymerization
00:36:01.25 don't have such filaments associated with them that
00:36:05.24 will average up in this way. So what this suggests is that
00:36:09.28 if you choose your protofilaments by an objective criterion
00:36:12.28 which suggests that they are under some kind of stress or strain
00:36:19.10 that is keeping them from going to be just like depolymerization
00:36:23.07 and then they are not polymerizing, you can
00:36:25.03 then find protofilaments that are there.
00:36:27.17 And our interpretation of this is that
00:36:30.07 these kinetochore fibrils are exerting force on
00:36:34.15 the bending protofilaments so that as the protofilaments try to bend, they stretch this fiber
00:36:40.26 and exert tension on the chromosome itself.
00:36:43.03 So this is a different kind of coupling,
00:36:46.07 one that does not involve rings,
00:36:48.10 one that need not be a motor, but that could simply be a static link of some kind.
00:36:53.14 Here is a drawing in which I have represented
00:36:55.26 protofilaments based on the tracings that I have
00:36:58.07 done of the microtubules themselves,
00:37:00.06 Protofilaments, sorry, kinetochore fibrils
00:37:03.15 that connect the protofilaments up into the chromatin
00:37:06.19 and this now is a simulation done by Grishchuk, Ataullakhanov and his students
00:37:12.23 and it is showing that even with a load
00:37:15.20 of about 40 piconewtons pulling in this way,
00:37:19.05 if you have tightly binding kinetochore fibrils,
00:37:22.13 that interact with polymeric tubulin, so that they will
00:37:26.19 stick to these bending protofilaments, but be released
00:37:29.14 as soon as tubulin falls off from the end,
00:37:32.26 you can make a processive motor that works perfectly well.
00:37:36.05 And indeed, some of the details of this motor that are now
00:37:39.05 described in the Cell paper that came out this year
00:37:41.27 give us confidence that this has advantageous properties
00:37:46.18 that may be even better than a ring for serving as a coupling
00:37:50.04 even though it is an improbable idea.
00:37:54.04 Structurally then fibrils are found in many places, but
00:37:59.22 we'd like to know what they are made from.
00:38:01.26 If we don't know the protein composition of these
00:38:03.24 structures, it is very difficult to do experiments that will
00:38:07.07 tell us definitive things about mechanism.
00:38:09.06 We have a number of ideas of what they could be
00:38:12.06 of course, because we have seen
00:38:13.21 that kinetochore related motors, like kinesin 7's and 8's
00:38:19.05 are both fibrous in their structure, and they can do the
00:38:22.23 coupling job. There are also non-motor proteins,
00:38:26.17 which are fibrous, that are localized
00:38:28.19 to the kinetochore, and NDC80 is perhaps the most attractive of these
00:38:32.23 because it is found across all cells where it has been sought,
00:38:38.02 and in every cell where such experiments have been done
00:38:41.09 and if you delete this motor, the chromosomes simply cannot attach to the spindle.
00:38:45.19 So NDC80 is an important fibrous molecule involved in
00:38:49.26 attaching chromosomes to spindle fibers.
00:38:52.16 It could well be part of this attachment machinery,
00:38:56.29 however, the evidence that now exists about NDC80 shows that it binds to the outside of the microtubule
00:39:03.01 and the way I have shown you the fibrils,
00:39:05.17 with the bending of the protofilaments, it almost looks as if the fibril attaches to the inside of the microtubule.
00:39:12.16 There are very few studies that have identified proteins that would bind to the inside
00:39:17.15 of a microtubule, so maybe NDC80 also has this function, or there may
00:39:24.06 be a new class of protein not yet identified
00:39:27.06 which can fulfill this function.
00:39:29.09 Or, alternatively, our resolution may not be good enough to give
00:39:32.23 the straight answer as to where the fibrils join the protofilaments.
00:39:35.26 And they may come around onto the far side and bind.
00:39:39.29 Certainly there are other fibrous proteins in the spindle. There are many
00:39:43.15 of them in the kinetochore, and so some of these might serve as connectors.
00:39:48.08 The molecular nature of this coupling is really something one wants to understand.
00:39:52.28 But it really is not yet known.
00:39:56.14 Evidence from localization of proteins
00:39:58.14 from genetic disruptions of particular components
00:40:02.08 from biochemistry, all of these different
00:40:05.01 kinds of experiments suggest that there are multiple factors
00:40:09.01 that are inolved in coupling chromosomes to microtubules.
00:40:13.00 Some more important than others, perhaps, like NDC80
00:40:16.02 but it may be that what we visualized in the electron microscope
00:40:19.16 is actually a little molecular zoo.
00:40:22.06 and there are multiple kinds of connections made by different components.
00:40:26.04 The fact is that this is a wonderful set of unsolved problems,
00:40:30.15 and it's a marvelous study or problem for future work.
00:40:35.04 It's the kind of thing where one hopes that many laboratories will come to partake
00:40:40.21 in this kind of research, contribute to
00:40:43.12 the knowledge that we have, because our group has been small.
00:40:47.00 We are enthusiastic about our work.
00:40:49.23 We have enjoyed the kinds of things we've done
00:40:50.27 as you can see from Katya's enthusiasm.
00:40:53.14 Fazly Ataullakhanov, the mathematician who has been responsible
00:40:56.21 for the supervision of these three mathematicians,
00:41:00.21 who are now becoming cell biologists as well
00:41:02.21 and we've had a wonderful time doing this work
00:41:05.13 but there's lots to be done, and we hope you and others will come and join us in this research.
00:41:10.27 Thank you. Goodbye.