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Session 7: Cell Division

Transcript of Part 1: Separating Duplicated Chromosomes

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

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

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