The goal of these three talks is to define the problems that a cell faces as it prepares for division and to describe some of the ways it solves them. In Part 1, both the length and amount of DNA are presented as problems for chromosome segregation, particularly in eukaryotic cells. The actions of cohesins and of chromosome condensation are described as solutions. The mitotic machinery is introduced, including its diversity of form across phylogeny, however, the features that appear to be conserved are emphasized. This lecture may be useful for upper level undergraduate and graduate courses discussing mitosis and eukaryotic cell division.
The second lecture describes some key experiments showing the dynamics of a formed mitotic spindle and the ways these may contribute to accurate chromosome motion. Experiments that reveal aspects of the processes by which chromosomes attach to the spindle are presented. Mitotic motors are introduced and discussed in the light of what they probably do and do not accomplish to effect chromosome motion, including acting to improve the accuracy of chromosome segregation.
The third lecture presents evidence, largely from McIntosh’s lab, that shows how microtubule depolymerization can move chromosomes in vitro and explores the nature of some of the protein complexes that can couple chromosomes to microtubules and take advantage of this reaction.
Separating Duplicated Chromosomes
Concepts: Cell division, chromosome segregation, mitotic machinery
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.
Do you see advantages for a cell in having its spindle form inside the nucleus, rather than in the cytoplasm? How about having the spindle in the cytoplasm and the chromosomes in the nucleus?
Understanding Mitosis through Experimentation
Concepts: Mitotic spindle dynamics, chromosome attachment to spindle, mitotic motors, chromosome segregation
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.
Moving Chromosomes to the Spindle Poles: the Mechanisms of Anaphase A
Concepts: Microtubule depolymerization, chromosome-microtubule linkages
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.
Richard 'Dick' McIntosh
Richard McIntosh is currently a Distinguished Professor Emeritus at the University of Colorado Boulder. He earned his bachelor’s and doctorate degrees from Harvard University in Physics and Biophysics, respectively. He taught cell biology at that institution briefly, then moved to the University of Colorado at Boulder, where he has worked ever since. His principal scientific… Continue Reading
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