Cellular Organization of Complex Cell Structures

Transcript of Part 3: Formation and Duplication of Centrioles

00:00:02.13	Hello. My name is Tony Hyman. I'm the director of Max Planck Institute in Dresden, Germany.
00:00:08.08	On the background, you see a movie of a C. elegans embryo
00:00:13.16	going into mitosis.
00:00:15.07	And, one of the things that strikes one, of course, when one looks at a movie like this,
00:00:19.20	is that the spindle itself has two poles.
00:00:24.18	You have a pole here, in red, and you have another pole over here.
00:00:29.06	And that's, of course, at the heart of all cell division,
00:00:33.17	because the chromosomes, when they decide, go to these different poles.
00:00:36.29	You have to have two poles in order for the chromosomes to segregate into 2 masses.
00:00:42.28	So, you can ask the question then, why are there two poles?
00:00:46.17	This is a question that's interested biologists for more than 100 years.
00:00:52.23	This is a picture from the work of Bovary, the great 19th century German cytologist.
00:00:59.07	And, this is from a book by E.B. Wilson, called the Cell in Development and Heredity,
00:01:03.29	where he summarized a lot of this knowledge
00:01:05.25	that was discovered around the turn of the 19th and 20th century.
00:01:11.10	And Bovary was also fascinated by this problem,
00:01:13.27	where you could see that the spindle always has 2 poles,
00:01:16.18	and how is this bipolarity set up?
00:01:20.16	What I'd like to talk to you about in this segment of this talk,
00:01:25.03	is the construction of a very complex protein complex
00:01:29.23	called a centriole.
00:01:31.19	Over here, you can see that centrioles are quite large on our scale.
00:01:37.06	Here we have our tubulin molecules, when we were making microtubules.
00:01:40.01	And centrioles are again another order of magnitude bigger, and therefore, complex,
00:01:44.16	in terms of thinking of their organization.
00:01:48.15	And, at least in some systems,
00:01:51.20	it's the way the centriole duplicates which defines the fact
00:01:56.21	that there are two poles of a mitotic spindle.
00:02:01.04	Bovary was also interested in this problem,
00:02:05.19	when he stained mitotic spindles with different dyes,
00:02:09.01	he didn't have access to fluorescence in those days,
00:02:11.28	but he was still able to see lots of substructure,
00:02:13.27	and you can see in this particular picture, which was actually taken by Joe Gall,
00:02:17.04	from an original microscope slide of Bovary,
00:02:20.03	you can actually see that he could see this little structure in the center of the centrosome,
00:02:25.07	which we now know is likely to be the centriole.
00:02:27.12	The centriole is in the middle of the centrosome.
00:02:32.20	The centrosome, known as the pericentriolar material,
00:02:35.21	surrounds this centriole, which tends to exist as a pair of linked centrioles,
00:02:41.19	which tend to be orthogonal to each other.
00:02:44.04	One of the questions that's always been interesting in that field is
00:02:51.03	how do centrioles grow?
00:02:53.24	It's fascinating that once per cell cycle,
00:02:57.02	each centriole makes a duplicated daughter centriole.
00:03:02.18	Just like DNA, you also make one copy of each DNA strand.
00:03:06.29	The same is true for centrioles, and that's therefore interested people very much
00:03:10.17	over the years.
00:03:12.04	And the work of, really, three people sorted this problem out
00:03:15.21	in C. elegans embryos, at a morphological level:
00:03:19.25	Thomas Mueller-Reichert and Eileen O'Toole, two electron microscopists,
00:03:23.22	and Laurence Pelletier, he was a cell biologist.
00:03:25.28	And they decided to attack this problem in C. elegans embryos.
00:03:29.20	Now, the problem with studying centrioles are
00:03:33.07	they're extremely low abundance -- there's only about 2 per cell.
00:03:36.24	If you take ribosomes, there are many 1000s of ribosomes per cell.
00:03:39.19	The biochemistry is extremely difficult.
00:03:42.13	And, they also change through the cell cycle.
00:03:45.15	So, most large complexes so far studied,
00:03:48.13	by structure, have normally been isolated biochemically - proteasomes or ribosomes.
00:03:53.26	So, how are we going to study this problem of centriole growth?
00:03:57.02	Well, let's ask a simple question first.
00:04:01.13	What I've done here, and you're going to see this through this talk,
00:04:04.21	is I've outlined the timeline of the cell biology of C. elegans on this axis.
00:04:11.11	And you can see the different events here, and you can see the time on this bottom axis here.
00:04:15.11	So, during this process, we can ask the question, when do centrioles duplicate?
00:04:21.02	Now, centrioles are small, and we can't see them by light microscopy.
00:04:26.14	We certainly can't see... well, we can see individual centrioles,
00:04:29.19	but we can't see very easily if there are 1 or 2.
00:04:33.28	And so, in order to do this, you really need to use electron microscopy.
00:04:38.15	So, then you want to ask the question,
00:04:40.17	during this time, when is it that centrioles actually duplicate?
00:04:46.17	And, in order to do that, you need to say, alright, here I am
00:04:50.03	at this time point X. What I want to do is look at the centrioles by electron microscopy,
00:04:54.20	and that's what we know as correlative light microscopy and electron microscopy.
00:04:59.18	We use light microscopy in a living organism
00:05:02.23	to get the timing of the system,
00:05:05.11	and then we have to go by electron microscopy
00:05:08.08	and look at that system and ask, what did the actual centrioles look like?
00:05:11.27	And a way you can do that is by using fixation.
00:05:17.20	But, we need to be able to fix at certain timepoints.
00:05:20.25	And we can do that, due to a nice little trick in C. elegans,
00:05:24.01	which is the embryos have an egg shell.
00:05:25.25	Now, this egg shell has been beautifully evolved over many millions of years
00:05:30.24	to keep everything out. These embryos exist in soil,
00:05:35.28	as far as we know, and they have to be able to resist any outside insults.
00:05:43.01	But, you can actually penetrate the egg shell with a laser.
00:05:48.11	You can take a laser beam, in a very space age experiment,
00:05:50.25	you can shine it on the eggshell and pop a little hole.
00:05:53.29	So, you can do this nice experiment
00:05:56.08	where you can surround the embryo in glutaraldehyde,
00:05:58.26	which is a fixative, and it doesn't go across the egg shell,
00:06:02.00	because it's such an amazing structure.
00:06:04.05	Then you pop a hole in the eggshell, the glutaraldehyde goes in,
00:06:07.20	and fixes the embryos. Let's look at that in this movie.
00:06:10.14	What you're going to see is the embryo move a little bit --
00:06:12.17	that's where you pop it with a laser,
00:06:14.20	and you'll see it fix. So, here it goes.
00:06:18.07	
00:06:21.03	Pop! You see it's fixed.
00:06:22.25	
00:06:25.25	And, pop! The other one's fixed.
00:06:27.09	And did you notice how, when we fixed it,
00:06:29.14	all the movement stopped. It's a very quick process, fixation.
00:06:33.12	Glutaraldehyde is a very small molecule, goes in, fixes it, then you can process the embryos
00:06:39.03	for electron microscopy
00:06:42.00	by serial sectioning.
00:06:45.15	And, what that experiment showed is that
00:06:50.02	centrioles are unduplicated about here.
00:06:54.14	And, if you look a few minutes later, they've now duplicated.
00:06:58.20	So, by metaphase, they've actually duplicated.
00:07:02.00	So, that's a very fast process, the centrioles have gone from unduplicated to duplicated.
00:07:07.09	And so we can conclude, then, that there's a duplication process that happens
00:07:13.05	early on in the cell cycle of C. elegans.
00:07:15.21	But then you can ask the question,
00:07:19.00	well, how do they duplicate?
00:07:21.20	And the technique we were using then--glutaraldehyde fixation--
00:07:25.26	was not good enough to tell us how the centriole is itself being made.
00:07:30.17	We were able to see unduplicated centrioles, and we could see these nicely formed centrioles,
00:07:35.00	but we weren't able to pick up the different stages of centriole duplication.
00:07:39.05	How do they form?
00:07:40.16	They're very complex structures, and that's the question we wanted to ask.
00:07:44.10	Now, in order to do that, we had to move to a different kind of technique,
00:07:50.05	which is known as electron tomography.
00:07:53.17	In order to do tomography, we needed to be able to come back and stop the embryos,
00:08:00.25	but we needed to be able to stop them by freezing.
00:08:02.23	So, one way that we preserve structures in biology
00:08:06.26	without disturbing their ultrastructure is by freezing... very fast freezing
00:08:12.05	preserves biological components without disturbing them as much in the ultrastructure
00:08:17.01	as does the fixation.
00:08:19.02	And what you do, is you freeze at very high pressure and then,
00:08:23.22	high pressure prevents the formation of ice crystals,
00:08:25.21	and then you can infiltrate the fixation at very low temperatures,
00:08:30.19	and that's known as high pressure freezing
00:08:32.04	and that's a way to preserve the ultrastructure of the system.
00:08:36.07	So, what we needed was a way where we could freeze the system,
00:08:40.12	in a time-resolved manner.
00:08:45.01	And so, we came up with a particular way of doing that,
00:08:48.23	using little tubes that you use for kidney dialysis,
00:08:51.14	you can suck embryos into them, you can follow the development
00:08:56.02	of the embryos under the microscope,
00:08:57.26	and then you freeze them in the high pressure freezing machine,
00:09:03.14	and then you process them for tomography.
00:09:06.10	Now, the problem was, when we started this experiment,
00:09:09.14	it wasn't easy to actually freeze them
00:09:14.00	at the time that one's interested in.
00:09:15.19	So, we used a new machine, developed by Leica
00:09:19.23	which allowed us to do time-resolved tomography,
00:09:22.03	and I'm going to show you this machine in action here.
00:09:24.22	What we've done is we've taken the embryos, we've put them into a tube.
00:09:28.12	We found out they're just at the right size, and at the right stage.
00:09:31.28	We put them into the high pressure freezer, and now we're going to freeze them.
00:09:35.09	So here it goes.
00:09:36.01	We're going to bring our hand in, and we're going to push it in to the freezer,
00:09:40.22	and then POOF, it's now frozen.
00:09:44.01	So, we rapidly freeze the embryos.
00:09:46.16	Now we can take them, and we can process them for tomography.
00:09:52.03	Now the key thing about tomography is that you look at very thick sections.
00:09:56.29	So, normally, in a standard electron microscopy, you look at 50 nanometers,
00:10:01.02	but you can look at 300nm sections, and you get a 3D picture of the way it looked.
00:10:06.01	I won't go into that in detail in this talk. You can find it elsewhere if you're interested.
00:10:09.27	But it's a way of looking at a 3D picture by electron microscopy.
00:10:14.05	So, we can look at centrioles at metaphase, and we can see how beautifully they look.
00:10:20.13	You can see over here, for instance, a very nice picture of a centriole
00:10:24.13	with microtubules around the outside.
00:10:26.17	And then we can see one over here.
00:10:30.00	So, what we want to do is look at these in these tomographic sections.
00:10:35.05	And, as I mentioned, one of the main tools for a cell biologist to link phenotype to structure
00:10:41.02	is electron tomography.
00:10:42.29	It's a way that we can actually go in and get at high structural resolution the way things look.
00:10:50.03	An in situ phenotype.
00:10:52.26	So, the problem is that we do genetics and we get a phenotype,
00:10:56.13	we want to know how that's changed the ultrastructural level,
00:10:58.25	we generally can't isolate them from the cell and look at them.
00:11:01.07	Rather, we have to look at them in situ.
00:11:03.26	So, what I'm going to show you now is an electron tomogram
00:11:07.23	of 2 centrosomes and centrioles early on, after duplication.
00:11:15.01	So, what we're going to... We're stepping through the section.
00:11:18.07	You're going to see, we're looking at one centriole
00:11:20.06	with its microtubules, then we're going to step through further.
00:11:24.22	Then we're going to come to the other centriole pair. They're about a micron apart.
00:11:28.09	And you can see what we've done there, in that tomogram,
00:11:31.10	we've reconstructed both the distribution of microtubules
00:11:35.02	and the centrioles. And you can see there's a duplicated centriole pair at each spindle pole.
00:11:40.13	So, then, we said, now we're going to go back in high resolution,
00:11:46.05	and we're going to try to understand what are the intermediates
00:11:49.26	in making centrioles using our techniques.
00:11:52.04	And what we learned there was quite fascinating.
00:11:54.26	We learned that the initial step in centriole formation was formation of a central tube.
00:12:00.20	And you can see that here, by tomography,
00:12:02.29	and a cartoon next to it. This little tube is forming next to this centriole.
00:12:07.28	It doesn't have any microtubules around the outside yet. It's just a naked tube.
00:12:11.29	What happened then next was the tube elongated.
00:12:18.17	So, it's the growth of this tube from what's known as the mother centriole,
00:12:23.25	And so that's what we've learned so far, is that the centrioles duplicate by...
00:12:30.18	They separate into two individual components,
00:12:32.06	and then the daughter centrioles grow from the mother centrioles by elongation of this tube,
00:12:37.29	and then the next stage was very fascinating,
00:12:40.14	because we found that the microtubules then associate around the tube.
00:12:44.11	But, what we found was that the microtubules...
00:12:49.16	eventually there are going to be 9 microtubules all the way around the tube.
00:12:53.25	But, in intermediates in centriole formation, there are fewer microtubules.
00:12:57.27	So, in this case, there are 7 microtubules.
00:12:59.19	And also, they have intermediate lengths.
00:13:01.26	And you can see over here these hooks that we found around the inner tube
00:13:05.26	that seem to define in some way the nine-ness of the tube.
00:13:10.15	If you look at a number of different centrioles, you can see intermediate products,
00:13:15.26	so that somehow the microtubules are binding to the tube and forming this 9-fold symmetry.
00:13:21.08	Here's a cartoon of the process.
00:13:25.28	You can see the tube elongating, and the microtubules binding from the outside
00:13:29.11	growing, and forming the microtubules around the outside of the tube.
00:13:35.06	Now, we've made this cartoon with reconstructions, of course, of fixed material.
00:13:38.28	We haven't seen the microtubules growing,
00:13:40.26	but we've inferred it by looking at many different particular specimens.
00:13:46.14	So, what we learn from that is that centriole assembly
00:13:51.08	proceeds through structural intermediates.
00:13:53.07	You have a tube. The tube grows over about 8 minutes,
00:13:57.14	microtubules associate with the tube over about 2 minutes,
00:14:01.01	and the mother centriole then matures during this process. I didn't discuss that
00:14:06.01	in the tomograms, but the mother centriole changes slightly during this process.
00:14:12.01	So, what was exciting about that discovery was we'd shown that centrioles
00:14:19.21	have almost a virus-like assembly, where they have structural intermediates
00:14:23.22	you can define by looking at the growth of the centriole itself.
00:14:29.12	But what we wanted to do next then, was to say, now what we want to do
00:14:34.14	is find the genes required for that process.
00:14:37.07	That's the morphology... what is the genetics?
00:14:39.28	What are the genes required for that particular process?
00:14:42.08	And, that turned out to be something which was relatively straightforward
00:14:48.10	using our RNAi screen, because of the work of my PhD supervisor, John White,
00:14:54.15	and a post-doc in his lab, Kevin O'Connell.
00:14:56.22	And to do that, you have to understand a little bit about the biology of a C. elegans embryo.
00:15:03.22	Have a look at a wild-type. You see the blue centriole pair.
00:15:07.25	They come in with the sperm. Now, they then separate, and each pole gets one centriole.
00:15:16.03	But, the centriole is duplicated, so you have a centriole pair at each pole.
00:15:20.01	Do you see that? The blue centriole... the sperm has brought in its centriole pair.
00:15:25.02	It's separated, so you look at each pole,
00:15:26.18	and you'll see that it has one blue centriole from the sperm,
00:15:29.18	and the orange one represents the duplicated centriole that duplicated
00:15:34.10	during the process of preparing a spindle, as I showed you in the early part of the talk.
00:15:39.17	And then you look at the two-cell stage... the same thing happens again.
00:15:43.17	Let's see what happens if you prevent duplication of the centriole.
00:15:48.24	What happens when you do that is a very interesting phenotype
00:15:52.01	because the sperm brings in a centriole pair, the RNA interference
00:15:57.00	for reasons we don't really understand doesn't work very well in the sperm
00:16:00.03	So, that's unaffected. And then, the centrioles separate, and one goes to each pole.
00:16:06.06	It turns out, you don't need duplication to form the pole.
00:16:10.04	So, if you don't duplicate your centriole, it doesn't affect the mitosis.
00:16:14.03	But, the problem comes in the next cell division,
00:16:16.24	because then each cell only has one centriole, not two,
00:16:20.18	and now it only makes a monopolar spindle.
00:16:25.24	So, normally instead of bipolar, it just makes a monopolar spindle.
00:16:29.05	And I'm going to show you some movies of that.
00:16:33.01	So, here is a centrosome duplicating at the end of
00:16:39.12	cell division, and you can see it moving into two different centrosomes.
00:16:43.19	It's duplicated, and at the two-cell stage, you have two centrosomes,
00:16:46.23	and you've got the chromosomes, which I've shown you there as well.
00:16:50.27	So that movie has both labeled centrosomes and labeled chromosomes.
00:16:54.00	Now, let's see what happens when centriole duplication is failed.
00:16:57.09	Well, everything is looking fine at this point.
00:17:00.07	We've made a spindle, it's all divided.
00:17:02.19	But what happens to the spindles at the two-cell stage
00:17:05.19	is these beautiful little half spindles will form without a second pole.
00:17:09.26	A really gorgeous phenotype.
00:17:12.17	I can't stop looking a those... they're just so beautiful.
00:17:17.29	And, in fact, what we then did was to go back to our RNAi screen and say
00:17:22.08	how many genes are required for centriole duplication?
00:17:27.20	You can take the 800 genes required for cell division.
00:17:29.22	You can rescreen them by fluorescent microscopy,
00:17:32.00	and you can look for ones that have that phenotype.
00:17:33.27	And from that screen, it turned out that we now know there are 5 genes
00:17:38.09	required for daughter centriole duplication.
00:17:41.01	So, in the end, also quite simple, there's not many proteins required.
00:17:44.28	You would think, wow, that's a complex process.
00:17:46.23	Doesn't that require a lot of genes? But, no!
00:17:48.07	It seems like these 5, as far as we can tell, seem to be sufficient.
00:17:52.19	Now, the analysis of these genes and a detailed characterization
00:17:59.21	was published in a number of different labs,
00:18:01.03	and I've illustrated some of the papers over here.
00:18:03.10	And what all of those studies showed was the same thing.
00:18:07.06	If you remove the function of any of these genes, you get a monopolar spindle,
00:18:11.00	as I've shown over there in the fluorescence.
00:18:12.25	And if you then do electron microscopy, you then prevent centriole duplication.
00:18:17.14	So, that's quite interesting.
00:18:19.20	We've identified a set of genes that we know are required for centriole duplication.
00:18:23.05	But, always when you do a study like this, you have the same problem,
00:18:27.04	which is, how are the proteins themselves related to the structure of the process?
00:18:34.18	We've done two different experiments now.
00:18:36.01	I've showed you the structural experiments where we've shown how centrioles duplicate.
00:18:40.08	I've shown you the genetics which shows you how
00:18:44.00	we identify the proteins involved in that process.
00:18:46.24	But how are we going to link the proteins to the structure?
00:18:52.18	What aspect and which proteins are required for which aspect of building this structure?
00:18:56.16	So then we linked the two of them together,
00:18:59.01	and that's what's so nice about doing this time-resolved tomography inside the embryo
00:19:03.28	is we can now go back and look at the mutant phenotypes by tomography
00:19:08.18	and ask how does that affect the duplication?
00:19:10.16	And when we do that, what we find is the following.
00:19:13.26	Here I've laid out again a timeline of duplication,
00:19:17.28	and also I've put at the top the proteins.
00:19:21.08	And as a little hierarchy of organization where there are two proteins Spd-2 and Zyg-1
00:19:26.13	which are required for all the other proteins to go onto the centriole.
00:19:30.11	Now, if you remove Sas-6 or Sas-5 from the cell, and then you do electron tomography,
00:19:37.20	you find no duplication either.
00:19:40.24	So, that suggests that Sas-5 and Sas-6 are probably required for forming the central tube.
00:19:46.21	But, Sas-4 was more interesting in its electron microscopy phenotype
00:19:54.24	because when we removed Sas-4, you still formed a tube, but
00:20:00.22	you don't form any microtubules around the outside of the tube.
00:20:04.23	So that tells us then that Sas-4, in some way,
00:20:08.13	is required for form the microtubules around the tube.
00:20:14.15	Let me just show you one tomogram.
00:20:18.02	of formation in Sas-4 RNAi embryos.
00:20:24.22	You can see that the mother is fine,
00:20:27.20	but the daughter only has a tube with no microtubules around it.
00:20:31.08	Can you see that here? That little purple...
00:20:32.28	The green is the mother, and the purple is the daughter.
00:20:36.25	So, that's what we conclude then from this study, which is that
00:20:43.01	the set of proteins forms onto the forming centriole,
00:20:48.00	and then we can show that Sas-5 and Sas-6 are apparently required
00:20:53.02	for forming the central tube, and Sas-4 is required
00:20:56.00	for forming the microtubules around the outside of the tube.
00:20:59.25	So, in that study, what I've tried to show you
00:21:02.22	is another very complex, intricate protein complex
00:21:09.21	forming from the arrangement of different molecules.
00:21:13.27	It forms an interesting structure, which is a different one from microtubules.
00:21:16.27	Microtubules are polymers.
00:21:17.28	This one seems to be a more virus-like assembly,
00:21:20.15	with steps of assembly process that we can isolate
00:21:23.01	And we can also find the genes required for it.
00:21:25.25	And we can show, in outline, how they're required for different aspects of centriole formation.
00:21:31.21	And the next stage, of course, will be to do more detailed structural work
00:21:35.03	to try and understand how the individual proteins affect, for instance,
00:21:41.03	the formation of the tube itself.
00:21:43.18	So, centrioles then, we believe now, form by a sort of virus-like mechanism
00:21:52.18	with steps in the assembly process.
00:21:55.06	And coming back to our scale, you can see that we've gone out
00:22:00.17	quite a few orders of magnitude now,
00:22:02.09	from our initial tubulin molecule, so we're actually looking at fairly complex structures,
00:22:07.05	which are a couple of orders of magnitude bigger than the molecules that make them up.
00:22:11.13	And so, you can see that slowly, we're putting the cell into subcompartments of organization.
00:22:17.15	We're not working on individual proteins, but they make these very complex structures.
00:22:21.22	Some of them more machine-like, say ribosomes, which make protein,
00:22:26.23	but other ones are more complex-like... polymers or like centrioles.
00:22:30.13	And by thinking about how these things are put together,
00:22:33.05	it helps us to understand the organization of the cell.
00:22:37.08	I'd like to thank... finish, by just... of course, the genomics itself is a very, very
00:22:45.09	time-consuming process involving many different people.
00:22:48.24	But, some of the key players are mentioned here.
00:22:51.14	As well as those involved in the centriole assembly.
00:22:58.02