Lessons From Yeast - Autophagy: Intracellular Recycling System
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| Lizenz | CC-Namensnennung - keine kommerzielle Nutzung - keine Bearbeitung 4.0 International: Sie dürfen das Werk bzw. den Inhalt in unveränderter Form zu jedem legalen und nicht-kommerziellen Zweck nutzen, vervielfältigen, verbreiten und öffentlich zugänglich machen, sofern Sie den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. | |
| Identifikatoren | 10.5446/45081 (DOI) | |
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00:00
Vorlesung/Konferenz
00:32
Vorlesung/Konferenz
01:13
Vorlesung/Konferenz
02:39
Computeranimation
03:18
Computeranimation
04:35
Vorlesung/Konferenz
05:06
Vorlesung/Konferenz
05:40
Computeranimation
07:52
Computeranimation
08:21
Vorlesung/Konferenz
14:36
Computeranimation
18:29
Computeranimation
20:45
Computeranimation
24:37
Computeranimation
25:58
Computeranimation
27:25
Computeranimation
30:39
Vorlesung/KonferenzComputeranimation
Transkript: Englisch(automatisch erzeugt)
00:14
Good morning, everybody. It's great pleasure for me to be here.
00:21
Since this is the first time to present our work in this excellent meeting, so today I will briefly mention how I launch into this autophagy field and also mention about recent,
00:41
our interest in autophagy in the East. Okay, this is my favorite organism. It is saccharomyces cerevisiae. I have been working on this tiny cell for more than 40 years.
01:01
Still I'm working on this cell. When I come back from the Rockefeller University in 1977, and joined to the Dr. Andraku's lab, and he told me, you can choose any research subject using yeast.
01:23
After a while, I decided to work on biofuel. It's not so many people interested in this organ, because it was sold just like a garbage dump,
01:41
in the cell. So not so many people interested in this organ, but as you know, plant cell has sold Rajbacchio. I thought that might be important for cell function. So I started work on the bacterial membrane,
02:01
and I found several active transport system on this membrane, such as amino acid, calcium, and other ions. And so, biofuel is not inert organ. It has a
02:23
important function for cellular homeostasis. And also, we found for the first time, a noble proton pump on this membrane with type ATPase. But, when I'm in 1988,
02:42
I moved to another college of the Tokyo University by myself, and I want to change my research subject. At that time, I was already 43 years old, but I want to switch to my subject
03:01
to another function of biofuel. I assume that biofuel might be the retic function in our cell, so just like lysosome in a membrane cell.
03:20
Before going to my work, let me introduce a little bit about the protein turn number within cell. This simple diagram shows the protein dynamics in our body. We are taking 60 to 70 to 80 grams
03:43
of protein every day from diet, but our body making 200 to 300 grams of protein every day. Just to these figures, strongly suggest our body is just a real state of their,
04:01
all, almost all of the, most amino acid for protein synthesis come from their degradation of the preexisting of our body protein. However, not so many people interested in the protein degradation at that time
04:22
because they thought it's not important for life, just a passive pathway. But just, I want to show one, just try to convince you that protein degradation is important.
04:42
This is rice field. Left panel, you can see there are green leaves making starch by sunlight. But if you see at harvest time in autumn,
05:01
all leaves turn yellow. What did that mean? All of their machinery for photosynthesis, chloroplast containing green pigment, completely degraded to amino acid, and those amino acid transported to the rice grain
05:23
to make our important protein for rice. So degradation, protein turnover is very important to generate next generations. So life is just a equilibrium state
05:42
between protein synthesis and degradation. And also in nature, starvation is the most frequent threat for life. So recycling system must be very important for survival.
06:05
Okay, intracellular protein degradation was interesting subject, but not so, not so easy to analyze. The first breakthrough was done by Christian DeGue.
06:22
He found lysosome as a ritic compartment in mammary cell. Single membrane organelle containing various kinds of hydrolysis. Then Roch-Ferrari's group, electron microscopist showed how cytosauric protein
06:46
deliver to the lysosome. In 1962, Christian DeGue coined the process as a delivery of their own cytosauric protein
07:05
to lysosome to be degraded. It's autophagy, self-eating process, meaning in Greek. Okay, since then, not so much progress.
07:22
Unfortunately, not so much progress was done about molecular mechanism of autophagy because at that time, electron microscopy is the only way to detect autophagy.
07:44
Okay, so there must be required quite good model system. Okay, let's move, go back to my work. This is a cell have large bioacetic compartment
08:07
and containing, it was known already bioacetic contains various kinds of hydrolysis. So if this is ritic compartment in each cell,
08:21
the problem I had is the simple when and what and how cytosauric constituent is delivered to back here to be degraded. Okay, as you know, if each cell face
08:44
nitrogen starvation condition, they start, quite dramatic cell remodeling, that's its population.
09:00
So this dramatic change inside the cell should accompany their massive protein degradation. So nitrogen starvation might be a good condition to work with. And also, as I said, I was working so long time
09:22
on the yeast vacuole. I knew vacuole is quite easy to see under a light microscope. And vacuole contain just a sole solution, a quite small amount of protein. So I knew if some structure get into the vacuole,
09:45
I can easily detect. So I tried to see what happened under light microscope during early step of starvation condition,
10:02
but I couldn't find any interesting structure. But I thought if I use a protein deficient mutant to see what happened under starvation condition.
10:20
Okay, this movie is exactly the starting point of my work. As you see, and this is three hours later, yeast vacuole contain so many just randomly moving spherical bodies.
10:44
I thought this is quite interesting phenomena. And this is a time course of this phenomena. After 30 minutes, you can see these structure in the work using vacuole, and they accumulated up to several hours in the vacuole
11:03
as shown in previous slide. So we started, Mrs. Baba and I started the electron microscopy on this phenomena. You see vacuole contains spherical bodies
11:22
and high magnification image here. You can see those structures, single membrane bound organelle containing just same density of liposome and various kind of site, plasmic structure within.
11:41
And also we found quite unique membrane structure next to the vacuole, just end up in a portion site, so. And double membrane structure in the site, so that is east autophagosome.
12:01
And this double membrane structure fused with vacuole. And you can see here the outer membrane of the autophagosome fused with vacuole membrane. And you can see quite unique membrane structure within vacuole.
12:22
Those are autophagic, we called autophagic bodies. Those are structures used previously, so in movies. So from this analysis, we draw this kind of scheme
12:40
on the autophagic process in yeast. If yeast cell face various kind of starvation condition, suddenly small membrane appear and enclose a portion of site, so form a double membrane structure.
13:02
And those structure target to the vacuole, and membrane fusion occur in the membrane structure, get into the vacuole. In wild-type cell, these structures are so immediately disrupted,
13:21
so nobody found this kind of structure without using popular protein as the efficient mutant cell. And also, the reason I show this figure, you can see the intermediate structure, and also you can see mitochondria here and here and here.
13:46
So autophagic degradation is quite unique. If compared to the ubiquitin protosome system, it's suitable for bulk degradation
14:04
of the site's component. Rhizome is quite large supermolecular structure, but you can see one autophagosome can degrade thousands of ribosomes,
14:21
and also, as you see here, mitochondria organ can degrade at once by autophagy. So next, obvious next step for us is a genetic dissection of this process.
14:40
So we want to have autophagy-defective mutant, but we didn't know anything about the phenotype of the autophagy-defective mutant, so we started just a morphological screening to get autophagy-defective mutant.
15:05
One graduated, Miki Tsukada succeeded to isolate the first autophagy-defective mutant, ATG1 mutant.
15:20
This mutant is apparently defective in the bulk protein degradation, not the starvation condition. However, that mutant has no phenotype in a very rich growth medium, but we found one phenotype of the ATG1 mutant
15:45
that showed this slide. An ATG1 mutant cannot survive after wrong nitrogen starvation condition, shown here. We assumed that might be caused by the defect in autophagy,
16:02
so we took this autophagy-defective mutant loss of viability as a first screening, and then microscopic screening, we got about 100 autophagy-defective mutant.
16:23
Genetic analysis, we could get 14 ATG genes. Since then, we proposed 18 ATG genes are essential for autophagy yeast, so first screening was quite efficient.
16:42
Obviously, next step is what is ATG gene, and we started cloning of ATG gene, that's the sequencing and the identification of ATG protein, but we found those ATG genes are essential for unique membrane dynamics
17:02
of autophagosome formation in yeast, but all genes identified is a new, novel gene, and we couldn't get anything from sequencing,
17:23
showing about function of those genes, but I moved to another institute, NIBB, and suddenly we could get many things about ATG,
17:49
and as shown here, about half of the ATG genes are involved in these two ubiquitin-like conjugation system,
18:02
ATG-12 conjugate to the ATG-5, and ATG-8 is conjugated, not protein, to the one of the phosphorylipid, phosphoryl ethanol aminbir amide bond, and those two conjugation system
18:24
are essential for autophagosome formation, and we found TOC kinase is an upstream regulator of autophagy, if we introduce lipomycin to block TOC activity,
18:46
exactly the same phenomena occur within rich medium, and those process are mediated by the phosphorylation ATG-13, and form a initiation complex of the ATG protein
19:03
to activate ATG-1 kinase. From those kind of analysis, we, within several years in NIBB, we could divide 18 ATG genes,
19:20
which is essential for autophagy, into these seven or six functional group, ATG-1 kinase complex, ATG-9 sole membrane protein, ATG-12-2-18 complex, and specific pH-3 kinase, and two conjugation system as I talked.
19:47
And fortunately, I was in the NIBB, my colleagues studied the ATG genes in mammals and plants, and we found almost all,
20:02
most three ATG genes are well conserved from yeast to mammals and plants. So, identification of ATG genes, quite quickly changed the landscape
20:20
of autophagy research in all over the world. My colleague, Nobolu Mizushima, first generated GFP-LC3 transit mouse. Using this mouse, we can see easily
20:40
where and how much autophagosome is formed in various condition. From those cells, we can see fibroblasts and just for the one hour starvation condition, you can see lots of autophagosome
21:02
are formed within cytoplasm. And also, Nobolu studied the maker, knockout mouse first, and he found autophagy is essential
21:21
for survival after birth. Many, many labs started work on autophagy using ATG genes as a tool, and now we know autophagy is not
21:40
just a starvation adaptation, it is very important, interest of clearance of the harmful protein and also elimination of invasive bacteria. And it's also important for the artist development stage of the fertilized egg.
22:03
And many people interested now, autophagy is relevant to various kind of diseases, especially neurodegenerative disease and cancer. And also, many reports said autophagy
22:21
is important for longevity and antigen presentation and so on. I do not go further detail on this, but now we know there are two major role of autophagy.
22:43
One is, of course, nutrient recycling, that's very important for starvation. And another function of autophagy is elimination of excessive or harmful materials from cytoplasm. And the selective degradation of various kind of protein
23:04
and organic and structure are very important to various kind of physiological homeostasis. And this slide showed the increase of the publication about autophagy.
23:24
I started autophagy work here only 20 or small paper appeared all over the world. And we found the ATG genes here. But now autophagy field become
23:43
so hot field in cell biology. But I want to emphasize even this kind of quick establishment one field,
24:01
but you can see it takes several years to become a really hot field. So I want to, okay, it's very important for young generation, you can start this so popular field of autophagy,
24:20
but also I want to say young generation challenge the not so popular subject. Okay, in the rest of my talk, I will mention about recent our interest in autophagy.
24:42
So we have so many fundamental question to be answered yet even in this autophagy. We are now working on when and what and how cytosolic constituents are degraded
25:01
via autophagy more quantitatively. So we need more about induction condition, various mode of autophagy, and what is the target of autophagy degradation. And it is important to understand
25:22
the final degradation product and the effect into the cellular metabolism. Just I want to show recent our work. Zinc is one of the essential iron
25:40
for every types of cell. We found zinc starvation in this autophagy and zinc under zinc deficiency condition, ATG mutant cannot grow further. And also zinc starvation induce massive bulk degradation.
26:06
And also another example is the each cell undergoes diox shift under glucose growing condition. And we found during diox shift,
26:21
again autophagy is induced. But ATG mutant cannot undergo proper diox shift.
26:41
The reason was deficiency of iron, e-iron. So if we add iron, ATG mutant can grow normally. So that means autophagy protein degradation
27:00
is important, not only protein degradation. Protein degradation is very important for iron homeostasis because iron availability in the cell increased by the degradation of those
27:21
that are iron-containing protein or organelles. And recently we found under starvation condition, nucleoside levels transiently increase in wild-type cell, but not in ATG mutant.
27:40
And we thought that might come from the RNA degradation. Tomoko Kawamata in my lab worked hard on this process, and we found responsible RNAs produce three prime XMP and turn to the nucleoside.
28:04
Nucleoside immediately transferred, converted to base and nucleolibose, and almost all base come out from the cell.
28:22
So RNA degradation, be autophagy, is not a recycling system, recycling RNA. So now we're working on RNA degradation, be autophagy, by using RNA1, one deficient mutant.
28:45
So under starvation condition, accumulate RNA, and what RNA transported to the molecule under starvation condition. We found there are several selective messenger RNA
29:07
selectively transported to the molecule, be autophagy. And also, it was my long-term hope,
29:21
we want to isolate autophagic body, and recently we succeeded to isolate autophagic bodies, shown here, so we can directly measure what kind of protein really delivered to the molecule.
29:41
To be degraded by directory, and so now we are analyzing about the degradation of cytosolic protein, be autophagy. We saw bulk autophagy is non-selective degradation,
30:04
but now we know there are some preferential degradation of the several protein, be autophagy. That means cytosol, side-parametric protein, is not evenly distributed. There must be change of state within cytosol.
30:24
So those might be help to understand our knowledge about how cell are organized. And finally, I want to say thanks to the excellent former
30:40
and present lab members, nice collaborators, supportive family, thank you for your attention.