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My name is Kai Simons, and I come from the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden.
Now we give three iBioSeminars. They will all be about lipids as organizers in cell membranes.
Cell membranes. They are two dimensional solutions of oriented lipids and proteins.
The matrix is made of the cell lipid bilayer. Cell membranes are made out of lipids.
The matrix is made from the lipids. And then there are proteins embedded in the bilayer.
Now in the last two or three decades, the proteins have received most of the attention.
One third of the genome, of our genome, encodes for membrane proteins embedded in the bilayer.
And many proteins, other proteins, spend part of their lives on either side of the membrane interacting with the membranes.
So many of the functions in our cells are membrane localized.
Now the lipids have more and more come into the background.
But in fact of course, they are as important.
Membranes are made out of proteins and lipids.
So take the lipids. We have the glycerol lipids, sphingolipids, and sterols.
And these if you look at them more closely. Then take the glycerol lipids.
They are very complex. So you have the glycerol molecule. You have the head group.
And then you have the two fatty acids.
So over a thousand individual lipid species can be analyzed.
And if you look at this complexity we see that there are several head groups.
There are these fatty acids in the Sn1 and Sn2 position.
They are of different length. They all have different numbers of double bonds.
And they can also be linked to the glycerol with ester or alkyl ether or alkenyl ether linkages.
So this makes this whole complexity. Also the sphingolipids are complex.
They are not based on the glycerol. They are ceramide based.
We have a sphingosine with the fatty acid amide bond to the sphingosine. This is the ceramide.
The hydrophobic backbone of these lipids, and then we have head groups.
Phosphocholine, like we have in phosphatidylcholine of the glycerol lipids, but also many, many glycan head groups
like you see here, sulphatide, ganglioside M3, or the Forssman glycolipid.
Now the big problem has been how to analyze this diversity.
So many labs still use very simple methodology: TLC, thin layer chromatography, or liquid chromatographic methods.
And they can only separate the classes, like sphingomyelin, phosphocholine or phosphoethanolamine.
But in fact now with mass spectrometry we can start to look at the full diversity of the lipids.
So in the 1990s, triple quadropole mass spectrometers came into play.
Today we can analyze lipid species, and we have there then the lipid species.
This just means that we can't differentiate between the fatty acids, but now with the new generation of hybrid mass spectrometers
we can look at molecular species, meaning we are analyzing the full diversity.
So in our institutes we have Andrej Shevchenko developing a shotgun lipidomics platform which now can analyze the full lipidome of cells.
The sample has to be extracted. We add internal lipid standards.
We infuse them into the mass spectrometer and then there are software programs
to quantitatively analyze the lipid profiles of cells, tissues, and organelles.
Now if you look at the lipid quantification then you see here you get all of these peaks.
And we add the standard internal standards and in this way we get quantitative lipidomes that you will see in these lectures.
So cellular lipidomes may contain up to 7000, we don't really know, lipid species.
It is like the genome. We thought at first that there were a hundred thousand genes,
and today you know it is only one fourth of that number.
So the full number of all lipid species is not yet known.
And lipids are important. And the question is what is this complexity good for?
Why do we need all these lipids?
Why do cells have to synthesize so many lipid species, metabolize them, and regulate them?
Well, lipids are important in forming the shapes of membranes, and therefore they are important for cellular architecture.
They are also important in signaling. You can hydrolyze away parts of lipids,
and then they signal into the cell as a part of the whole signaling transduction system have at their disposal.
They also regulate the function of membrane proteins.
Protein-lipid interactions is a big forthcoming area which needs a lot of work.
Now you have also the question of how these lipids are distributed in the cell.
Membrane trafficking for instance. Or the issue of how lipids are part of mechanisms to create subcompartments in cells.
So I will now in my 3 iBioSeminars, in the first one, this one, talk about the role of lipids
in organizing the biosynthetic pathway from the endoplasmic reticulum to the cell surface.
Second one will be about this membrane organizing principle which we call lipid rafts.
And the third iBioSeminar will be on the biogenesis of the glycolipid-rich apical cell surface membrane in epithelial cells.
So you know proteins are made in the cytosol in cells,
and then they have to be distributed to different destinations where they carry out their functions in cells.
And there is also a very important pathway starting in the endoplasmic reticulum
over the Golgi, endosomes, cell surface, all for export out of the cell.
Also lipids have to be distributed. So many of the lipids are made in the endoplasmic reticulum
and then moved from there to different destinations either by membrane trafficking mechanism or by other means.
And then we get the full lipid complement which is different in the different membranes surrounding the organelles in our cells.
So if you look now at the three major groups, sphingolipids family, the glycerol phospholipids, and also cholesterol.
Then cholesterol, let's start with cholesterol.
So cholesterol is made in the endoplasmic reticulum, but the concentration there is kept very low.
Cholesterol after synthesis is moved out towards the Golgi and to the plasma membrane
and to other organelles, and there is a gradient, low in the endoplasmic reticulum, low, a bit higher in the Golgi
and then highest in the plasma membrane.
So cholesterol is synthesized in the ER and then moved out.
Now what are the functions of cholesterol? So first of all cholesterol makes the bilayer more impermeable.
It is more robust then. Therefore you have more in the plasma membrane, which is protecting the cell.
Another function is that it thickens the bilayer.
The bilayer thickness increases somewhat with cholesterol in there.
Now one important question in this field is that you have now the bilayer with the lipids,
and then you have the proteins embedded there. So if you look at the proteins the parts that
are facing the bilayer are all hydrophobic.
And the parts, let's say the alpha helix here, it's a transmembrane domain of hydrophobic amino acids.
And very important is that the length of this hydrophobic domains have to match the thickness of the bilayer.
If hydrophobic areas are open to the outside aqueous solution, that is not tolerated.
So therefore the bilayer can extend as you see here, order, become thicker,
to accommodate the transmembrane domain, or the membrane domain can tilt.
The protein can tilt or it can be compressed to be covering the hydrophobic domain of the protein.
Now very important is that cholesterol depleted membranes, like the endoplasmic reticulum,
they tolerate, they are deformable, and therefore they tolerate transmembrane domains of different lengths.
But then if you increase the cholesterol concentration, then this mismatching becomes a problem.
And it is much more difficult to accommodate proteins of different lengths in a cholesterol-rich membrane.
This means that cholesterol induces protein sorting. So a small, if you have it, bilayer, that can accommodate these with cholesterol in them
then the short transmembrane domains will come together and be separated
from the long ones. And this is a principle that is used now in the trafficking from the endoplasmic reticulum
over the Golgi, to the plasma membrane. So the membrane is thickened towards the cell surface, which is the thickest,
and this induces now a sorting principle where you start now with the endoplasmic reticulum.
You put in proteins of different lengths. The endoplasmic reticulum membrane tolerates that,
then they come to the Golgi where the cholesterol increases
and then you see the following phenomenon.
Plasma membrane proteins that have to be transported further are longer in their transmembrane domains,
20 amino acids about, whereas the Golgi ones, as you see, are shorter.
So you have a biophysical principle of how you sort these proteins this way.
and this was work by Mark Bretscher and Sean Munro in the early 90s.
And it has now been confirmed in many ways.
Now thus we have a biophysical principle, transmembrane domain length,
cholesterol increasing in concentration, we get the sorting principle.
But that of course is not enough.
So let's now go to the sphingolipids. So the sphingolipids are made in the Golgi mainly.
So the endoplasmic reticulum is very low in sphingolipids, that is cholesterol and glycerol phospholipids,
and the sphingolipids start to be made, sphingomyelin and the glycolipids are made in the Golgi,
and then increasing in concentration, like cholesterol, towards the plasma membrane.
And here comes another function of cholesterol, and that is that cholesterol and sphingolipids like each other.
They associate with each other, and this leads to this principle of sphingolipid/cholesterol rafts.
So in the bilayer, if you have these, now these red lipids, ceramide based sphingomyelin
or the glycolipids then you see that these lipids, if you look at their hydrocarbon chains, they are all saturated.
They are straight, whereas the glycerol lipids have unsaturated fatty acids usually in the Sn2 position.
They are kinked. And so cholesterol likes to be under the head group of the sphingolipids,
associate with each other and pack more tightly,
segregate away from the unsaturated glycerol lipids.
Now this all happens in the outer leaflet of the bilayer.
The outer leaflet facing the outside if it's the plasma membrane.
Now also the inside of this assembly is more ordered, and here we don't know exactly what is the principle here,
but they come together and form this liquid ordered, more tightly packed domains.
And important here is this, that you see here, the sphingomyelin and the glycosphingolipids are more saturated.
The fatty acid is longer. And they are... they have hydrogen groups
so they can intermolecularly hydrogen bond and this assembly also becomes somewhat thicker.
So this now is a principle for segregation in the membranes.
And rafts function as a sorting principle because they not only contain lipids, the sphingolipids and cholesterol among others,
they also contain proteins, specific classes of proteins. We have the GPI anchored proteins, glycophosphatidylinositol anchored,
two saturated fatty acids sticking into the outer leaflet of the bilayer.
Or we have on the inside doubly acylated proteins that have two myristolates in the amino terminus and then a palmitoyl nearby,
and they also associate with this. And then we have transmembrane proteins which also like to be in rafts.
And many other proteins that are excluded from the rafts.
So rafts they come in as a sorting principle in the trans-Golgi.
Sphingolipids, cholesterol, and proteins are sorted and then transported to the cell surface.
Now let's look at this in more detail. And we shift now to yeast, Saccharomyces cerevisiae,
a very great model system for cell biology because it is simpler than most other cells.
So there are two pathways from the trans-Golgi network in the Golgi, the exit station.
One, the red one you see here, going to the surface, and then another one going to endosomes, and then to the surface.
And the idea is now that this red pathway, is it the raft pathway?
If we really think that these rafts are involved in the sorting process, then we have to get some evidence for this.
So the carriers that are formed in the trans-Golgi network that you see here, if you would isolate them
they should be enriched in sphingolipids and sterols.
And have much more than the donor compartment, than the trans-Golgi network.
So let's look at this. So now we want to isolate using immuno-isolation techniques
yeast secretory vesicles coming from the Golgi and carrying a raft transmembrane protein
which is called this FusMid cargo protein.
So now the idea is to isolate the vesicles formed in the Golgi carrying the FusMid cargo.
Okay, yeast is great because there are genetic strategies. Randy Schekman is talking about these in his iBioSeminar.
There are mutants for each step in the pathway, and you can use these to study questions of this sort.
And there are mutants, for instance the sec6 mutants, which block the delivery of these carrier vesicles on their way to the surface.
They accumulate in the cytosol. And now we express this cargo protein, the FusMid cargo protein,
and then we restrict the temperature, we lower the temperature, for these are temperature sensitive mutants,
and the we get this accumulation as you see of these red vesicles in the Golgi.
And now coming from the Golgi, accumulating in the cytosol.
And if you look at them, then one subset of this red vesicles are those that we want to isolate now.
Okay, so we homogenize the cells after we have accumulated the vesicles in the yeast cells,
and then we fractionate, and the final fractionation is a cell density gradient centrifugation,
and then we take the fraction in the gradient that contains our FusMid transmembrane cargo protein.
Now look at it. This FusMid protein has a GFP tag so we can see its color. It has a TEV cleavage site so you see, and it has nine Myc epitopes.
It's epitope tagged.
Now we have this fraction. We then add antibodies against the Myc epitopes.
They bind to it, and now we take a cellulose fiber immunoabsorbant,
which contains antibodies against the Myc antibodies.
And then we pull down the vesicles.
And when we have done that we now cleave off the vesicles using this TEV protease.
And what do we get? We get vesicles which look exactly like those that accumulated in the cell.
100 nm diameter. And now we want to analyze the lipid composition
of these immuno-isolated, very pure vesicles carrying our raft cargo protein.
Okay, so the yeast lipids are similar to mammalian lipids. They have sphingolipids, ceramide based,
but the head groups are a bit different.
They have inositolphosphate, that is the simplest one,
and you can add mannose, or you can add even one more inositolphosphate.
So we shorten this with IPC and MIPC and M(IP)2C.
Only three classes of sphingolipids. They don't have cholesterol. Instead they have ergosterol, which is very similar.
So these are the lipids we are looking at now. We look at the whole lipidome using our quantitative lipidomics platform
that we have built together with Andrej Shevchenko's group.
And now we can just extract the yeast vesicles that we have immunoisolated.
We add the internal standards. We infuse them into the mass spectrometer, and then we analyze.
Now we are now analyzing the molecular lipid species using hybrid mass spectrometers.
And if we look at the whole lipidome we see that there are two hundred molecular species consisting of 17 lipid classes.
And now let's look at the vesicles. We have also isolated the donor compartment
the trans-Golgi network, and now we compare them.
And what we see here is the full spectrum. Here. And if you look at the ergosterol,
it is the major component in the vesicles, and the other major component is down there, the MIP2C, the most complex sphingolipid.
So the vesicles are enriched exactly in those lipids that we hoped they would be enriched for.
And then you look here and you see that sphingolipids and ergosterol are over two-fold enriched over the trans-Golgi network isolates.
So indeed we have now shown that this red pathway is a raft pathway because we have ergosterol and sphingolipids enriched in these FusMid vesicles.
So we have seen lipid sorting. So in the process of making these vesicles we get more sphingolipids and more ergosterol in there.
Now we also used a visual screen.
A screen to look for genetic mutants which are somehow blocked in this pathway.
And the problem is this. Well, first of all we used the same cargo protein,
the FusMid-GFP, but only containing GFP, and not rest, not the Myc epitopes.
And important in this case is that there are two pathways to the surface, from the Golgi.
Now if you block one pathway, it turns out that the component which usually take this pathway take the other pathway.
It is like going to the station from here.
The road down the hill is blocked, then we take the other road, and we reach the station.
It takes a little longer, and if there is a real problem many of us in the city, we have a traffic jam,
but the traffic jam will in the end get the cars where they should.
This is the same thing in the cell. We used this traffic jam as a screen in principle.
That means now that in the wildtype, not in the mutant cells, we are deleting all the genes in yeast and looking at each deleted mutant.
And in the wildtype we see the protein reaching, the way we do the assay, the cell surface, as you can see here.
But in the traffic jam mutant you see inside accumulation. That is the traffic jam.
Well, looking at all the genes, we got 24 mutants, and very importantly, we got six lipid metabolism mutants.
And what were they? Well, they were two ergosterol mutants, in the ergosterol synthesis pathway, and four sphingolipid mutants,
exactly those lipids that we have now implicated in this transport pathway.
So if you look now at one of these, for instance, sur2, it is a hydroxylation mutant,
and this leads to missorting to the vacuole and to Golgi block.
And another mutant is elo3. It is a fatty acid elongation mutant and leads to missorting to the vacuole.
So small changes in lipid structure leads to traffic defects.
So in this case if you look here you see these hydroxyl groups are missing in the hydroxylation mutant.
And if you look at the elo3 mutant, you see down here, it is not a C26, long fatty acid, it is a C22. It is shorter.
And if you go now to the raft concept, what does it mean?
Well less hydroxylation means less intermolecular hydrogen bonding
so you do not have this association between the lipids in the assembly functioning as well.
And the long saturated fatty acid probably penetrates from the outer leaflet into the inner leaflet
and organizes it in this way. Now it is shorter, and it is not doing its job properly.
So you can see that these changes indeed lead to defects as we predicted if rafts are involved.
So now another prediction is that when you take the vesicles that are formed in the Golgi for delivery to the cell surface.
If they are containing rafts, the packing of the rafts should be higher.
That is also one of the issues as you remember.
Now how can we measure that? Well, we can take a probe; it is called C-Laurdan. It goes into the membrane
and it can feel how much water is in the membrane.
So if the membrane is disordered and not so tightly packed, there are some water molecules in the membrane.
But if it is more tightly packed, there is less water.
And you excite then in a... you take for instance these vesicles, and you add C-Laurdan, and now you excite at 800 nm
and then you read the spectrum. And if you have high lipid packing the emissions will be at 430 nm.
And if you have low lipid packing, it is 490 nm.
And if you do this, then we see that the vesicles are more tightly packed than the donor membrane, the TGN.
Again speaking for a lipid ordered change to more packing, higher packing, during this formation of the vesicles.
So we have now shown that, gone through with you some principles-
that lipids are involved in membrane transport and trafficking from the endoplasmic reticulum
to the Golgi and from there to the cell surface.
And we have seen that cholesterol leads to a sorting of proteins depending on their transmembrane domain length.
Short proteins stay in the Golgi, and longer proteins, longer transmembrane segments, go to the plasma membrane.
And then comes this principle of raft interplay with sphingolipids and sterols are sorted with proteins to the cell surface.
Now I must not forget to mention that there are, of course, other principles involved as well.
We are going through them here. For instance, the major principle of making a vesicle from a donor compartment
for delivery to the next compartment is by having transport signals in the cytosolic domains
of the cargo protein, which then bind to adaptors, cytosolic adaptors,
which in turn bind to coats, like clathrin coats, and in this way you form with other proteins a vesicle which then takes off.
And we are here thinking about another model where lipids come into play, where the raft concept leads to sorting of both lipids and proteins.
And in the final lecture, I will come back to this issue of how this mechanism works.
And here I'm going to end, and the next lecture will be on this dynamic subcompartmentalization concept,
the raft concept, and how it works in more detail.
And finally some acknowledgements, all of the people who were involved in the recent studies in my lab. Thank you.