Thank you.
I want to start by thanking Countess Bettina, Wolfgang Schürer
and the other members of the Council for the invitation to be able to come back and talk with other laureates.
And mostly to talk with the students and other young scientists here, which I always find very enjoyable.
So I'm going to give a slightly different talk, I'm not really going to talk about GFP.
I'm going to take the advice of a friend of mine, a colleague Stuart Firestein
who wrote this wonderful book a couple of years ago called "Ignorance: How It Drives Science".
Because he says in this that when two scientists get together they get really rapidly bored by finding out
what they’ve already accomplished, but what is really exciting is what are the problems that still stump them.
And what they are interested in at the moment.
So I'm going to give you a little bit of some of the questions that we’ve been interested in for a while.
And we have gotten some answers
but I hope I’ll be able to give to you some ideas about the some of the other problems that we are interested in.
The main problem I’ve been interested in for a very long time has to do with the mechanical senses.
For our senses in general there’s this basic question: how do we get signals from the world around us?
We’ve known for a 130 years about the molecule rhodopsin, so we know how vision works, how light is sensed.
To some degree we’ve known, as Brian Kobilka talked about, about receptors that detect chemicals.
So we know a little bit about chemical signalling but we have a large number of receptors,
or a large number of senses that work by mechanical manipulation.
I'm going to take advantage of an old picture of myself to point out some of these.
So we have hair cells in the inner ear that allow us to hear.
We have other hair cells there that allow us to detect acceleration.
We have cells in our tendons and in our muscles that allows us to understand the stretch of these organs.
We have numerous cells, in fact five different sets of cells, in our skin that allow us to sense touch.
And we have cells that detect blood pressure.
All of these have one thing in common.
We haven’t a clue how they work at the molecular level.
So it keeps me in business for quite a while.
And so when I was a postdoc I started working with John Sulston using C.
elegans to understand the sense of touch.
I want to tell you something about the colour code in my slides.
Anyone whose name is in red is someone from my laboratory.
Anyone whose name is in blue is a collaborator.
And anyone whose name is in black did something I wish I had done.
So if you take the animal here - we take a very sophisticated piece of equipment:
an eyebrow hair and glue it to a tooth pick.
We can tickle the animals in the head and they will move backwards.
Tickle them in the tail they move forward.
And animals that are missing these cells in blue no longer sense that touch and cannot move.
But they are not dead, because we can prod them and get them to move in other ways.
So these are the six cells that sense touch.
We take a cross section of the animal we find they have a very original, or unusual way of microtubules.
They have special extra cellular matrix and we have been able to mutate the animal, get well over 500 mutations
that make it touch-insensitive.
And we’ve cloned and mapped all of these genes now and all the mutations define 17 genes.
5 of them cause touch-insensitivity because the cells aren’t made.
They affect the development of the cells.
That’s been one of the overall questions we’ve been very interested in.
But 12 of them, the cells are made they just don’t work.
We hoped that these would give us some idea about the function as touch sensors which in fact they have.
So with regard to the determination of cell fate we'd like to know,
'How do these cells acquire the particular features that they do?'
A general question in developmental biology: How is cell fate determined?
We also wanted to know how that fate can be maintained.
Because it's not enough to throw the switch, the switch has to be kept on.
We’d like to know why there are only 6 of the cells in the animal that have the particular features
that these do, the microtubules etc.
And we'd also like to know some of the subtle differences among these cells.
For example these cells in the front have cell bodies with the single anterior process,
whereas the cells in the tail have the anterior process but also a posterior process.
There’s other subtle differences which I’ll talk about, which are about synapsis and so on.
So we'd like to understand how that takes place.
Over the years we’ve made some progress on understanding the cell fate determination.
So I’m going to give you a quick run through history.
In 1981 we discovered 2 of the genes affecting the development.
We found that the first one unc-86 was needed to make the cells that were going to become touch cells.
If they are not there the cells aren’t made.
Mec-3 is expressed in the touch sensing cells and without it they are unable to differentiate:
they don’t make the microtubules, they don’t make the extra cellular matrix, other things as well.
And so we had this model that unc-86 must be important for mec-3 and that mec-3 must then turn on subsequent genes in the cells.
It would be called a master regulator in terms of this.
Then in 1988 we finally cloned mec-3 and found that it was a transcription factor.
In 1989 we found the interesting fact that this transcription factor was needed to keep itself on.
So this question of maintenance and development required this as well.
We then found that unc-86, 4 years later, unc-86 actually bound to mec-3,
it not only turned it on but it worked with it for the subsequent differentiation.
So it wasn’t A turning on B turning on C but A turns on B and then A and B work together.
But it still took us 5 more years to actually show that.
And we thought, okay now we understand about development.
And then a little bit later, 2010, we discovered another transcription factor that was turned on,
and that this transcription factor was actually needed to keep the maintenance working appropriately.
It was sort of a cellular insurance factor to make sure the maintenance worked.
And we are still finding now new genes that we are adding to this.
But I wanted to point out, I want you to really look at the dates here, it really took a considerable amount of time.
We have some idea about why it is that there are 6 touch cells.
That is because there’s a whole combination of different transcription factors and other factors that are needed for this.
We’ve even made some headway into understanding how it is
that the cells are different from side to side and at the end of the animal.
Basically, because like in many organisms, there is a cluster of the Hox genes.
And so we thought, oh this is good, we’ll have 1 gene needed for 1 set, in fact these 2 genes are expressed
and are needed for the development of the tail.
Put them in the head cells the head cells become like the tail cells with the posterior process.
And we said, okay there’s got to be another one that is needed for the head cells.
And sure enough we found it. Except for one slight problem.
It's expressed in both cells, both types of cells.
Well we were sort of saved for this because we found a co-factor that was only expressed in the anterior cells.
Again a little more complex here. Now, let me summarise a lot of work.
This is a graph of when we had papers on various fabrics and you see,
just as in the example before, that nothing is really linear.
We really were working on one project then we shift to another project then many years go by
until we start to have an insight into it.
People sometimes, especially students, ask me, "How do you deal with the frustration of science?"
- which is a natural part of science.
And I say, "We work on more than 1 project at a time so we can avoid it."
So I strongly suggest that if you can work on more than 1 thing at a time you’re in good shape,
because usually something is going on that’s exciting.
One of those things of course is this thing, which is the paper on GFP in 1994.
But I want to say a little bit about the touch circuit.
Now, many of you may know that in 1986 there was a spectacular paper, a very short paper only 340 pages long,
that was published by Sidney Brenner and John White and others, in the Philosophical Transactions of the Royal Society,
which was the complete reconstruction of the nervous system of C. elegans.
Every gap junction, every chemical synapse and all of the three 302 nerve cells structure.
It really is quite a remarkable accomplishment.
It doesn’t mean we understand the nervous system and I’ll try to explain why in a moment.
But, I want to talk about the touch circuit.
At first we thought it was very simple.
The touch-sensing cells are sensory cells, so they get signals from the outside
and then they connect to other nerve cells.
That’s it. And we could use the circuit to try to find out what it was.
So we find out that the cells up in the head make gap junctions to interneurons
that go onto motoneurons and make the animal go backwards.
The cells in the tail make gap junctions to a different interneuron.
That goes to other motoneurons and make the animal for forward.
Wonderful!
Then another thing comes up, the cells in the head make chemical synapses
which we now know are inhibitory to the opposite set of interneurons.
And in the cells in the back do the same thing.
This actually produces a very interesting problem, I think.
The problem is, both sets of cells, the ones in the front and the ones in the back, recognise both sets of interneurons
and yet they make very different chemical connections: one a chemical synapse and the other is a gap junction.
How do these virtually identical cells 1) identify those cells and
2) how do they make connections to them that are distinctly different?
And we don’t know the answer to that.
But there’s been another problem that happens with this system.
It's that later 2 more cells go in, so we now have the 6 touch cells,
and they actually connect up to a completely different set of interneurons.
So there’s a modification in the development of the nervous system that we would truly love to understand.
And if this weren’t enough, we can see that there are a whole series of other connections that these cells make to other systems.
One, the one at the very tail there FLP-20, that’s a neuropeptide
that the cells release and that’s acting on some other cells in the male tail.
So this apparently simple system is incredibly complex.
And there’s obviously a lot of integration of sensory signals in this particular cell.
Now, I also said that we looked at the function and we cloned all of those genes.
I want to point out a couple of things that came out of these studies.
The first was that we identified a new class of sodium channels that are now called the DEG/ENaC series.
They also had unusual mutations in them that caused the channels to be open and caused the cells to die.
So these are arguably one of the first of the so-called channelopathies.
We discovered in MEC-2, working with Thomas Benzing who’s now in Freiburg,
that MEC-2 and similar proteins to it, including one in the human kidney,
are in fact membrane-localised cholesterol-binding proteins, a new class of cholesterol-binding protein.
We found in the MEC-17, we discovered a new gene MEC-17 that was needed for this function and others
then showed that it was actually the modification gene that put acetyl groups on alpha tubulin.
And finally we have now, we are doing some work to show
that another one of the proteins actually looks like a new class of ER chaperone.
Now, I'm basically as I say a geneticist and so when we try to address the question of the sensing of touch,
it actually was a very difficult experiment to do but we were really saved by genetics.
Bob O’Hagan, a graduate student, Miriam Goodman who has 2 colours because she was a postdoc
and then became a collaborator, she’s at Stanford, developed a nice method
to actually record from the cells which involves taking the animals, poking them to get rid of most of the internal pressure.
Because if they didn’t, then they put the electrode in, the animal would explode - makes the experiment a little hard!
Once you get the pressure out you have a little cut here, the cell comes out, you see it because of GFP,
you can record from it using a patch pipette.
And then you can stimulate the animal by touching it.
And when we did that, or when they did that, so here’s the pulse, they get a very nice drop here.
This is indicative of a sodium current which these channels are known to be part of.
Now if we looked at MEC-4 mutant that didn’t have the gene there was absolutely no activity.
Now, this is an experiment that a lot of people do in genetics.
They get rid of a gene to see what happens and that’s a very good start.
But there’s a problem with this.
That problem is you really have no idea how connected the gene product is with the results.
So let me give you an analogy.
If you were interested in what made a car move forward,
you would find that one essential component to make the car move forward is the door key.
Because if you don’t have the door key you are not going to be able to get the car moving.
But it's clearly very much removed from that.
But to drive the analogy a little bit further, what if you found a component
that you didn’t get rid of but in fact you just changed its shaped or you changed its form in some way.
And now every time you did everything that made the car normally move forward the car would move backwards.
Then you’d say that has to be a component that’s integral to this process.
And we had the genetic equivalent of that.
Because we found that when we had special mutations, missensed mutations in the two channel subunits, MEC-4 and MEC-10
and we looked not at -74 m/v but at +6 m/v, now there’s less of a driving force for sodium.
And so the wild type just has these small little blips but when we looked in the mutants,
if we looked in the mutants in this condition: no activity at all.
But at +6 m/v we get the activity except now the peaks are going in the other direction.
And the reason was we changed the channel from a sodium-selected channel,
so sodium rushes into the cell, to a potassium-selective channel, now potassium was leaking out of the cell.
And this was our indication that this was the sensor, turned out to be the way that most people have looked at this.
So now we have the sensor and we started thinking about, well you know, our sensors are not really on/off systems.
They get modified. Our sensors, we'd like to talk about the sensors as being isolated
but they actually have a lot of influences by others, other senses.
And so we were quite interested and I had a wonderful graduate student in the lab, Xiaoyin Chen,
he likes to be called Robert, a past person here at Lindau.
And he found these car speakers in the lab and decided to ask, what was going to happen
if he put the worms on the plates and vibrated them for some time.
So he set this up in the lab for several hours –
and during which time he left so that everyone else in the lab could hear this.
And then tested what happened when he took the animals off and tested their touch.
Now what we expected was that with continued vibration they would habituate,
they would become less and less sensitive to touch.
And that’s how it started.
But as he looked further they then became more sensitive.
Now interestingly this return to sensitivity only happened in the anterior cells not in the posterior cells,
only in one set of cells and not the other.
He then found 3 other ways of modulating touch, again over several hours and only in the anterior cells:
high salt, low oxygen, or the dormancy state called the dauer.
He went on to sort out what this was working with our channel, our MEC-4 channel,
and he found that vibration worked through integrins and focal adhesions serving as a secondary mechano-sensor.
And he worked out the pathway, it leads to ubiquitination or in this case the prevention of ubiquitination of the channel.
Whereas hypoxia and dauer activate the insulin-signalling pathway
and in fact hypoxia and dauer prevent the signalling from taking place.
And if this signal doesn’t take place now the channel gets ubiquinated and is taken off the surface.
Thus making the cells less sensitive.
And he found that a different set of cells used a different insulin to affect the same pathway.
So I want to say a couple of points about this work.
The first is it says that neural hormones, in this case two different insulins,
are really important for the circuitry, the modulation of these.
So these sensory cells are not just getting inputs from the outside,
they are getting internal inputs that are modifying their function.
There’s also modification in the cell.
And there are several points of integration of the sensory signal.
First in these cells, there’s 2 cells in the pharynx called M4 and I5 that release this insulin.
These cells integrate both of these 2 signals, hypoxia and dauer.
The insulin receptor integrates the effects of both of these insulin-releasing cells.
And further down here the AKT-1 product, the AKT kinase, integrates the effects of the insulin pathway
and this integrin pathway as well.
All of which leading to these other things.
So there’s many levels of sensory integration, both outside and within the cells.
One final thing, we were very intrigued about the fact that insulins are needed in the signals.
We have no idea whether there’s really a connection here but it turns out
that most people go into the doctor and then are subsequently found to have Type-2 diabetes.
Not because they have sugar in their urine or any other problem,
but what they’ve complained about is numbness in their fingers and toes.
So who knows if there is a similar regulation perhaps in humans regulating the amount of perhaps receptors on those touch cells.
We tried to figure out what this was, why this happened.
And I want to show you a couple of movies.
Showing some animals, you see this animal coming up in this first movie here.
These fungi have these lassos and somehow this animal just has to stick his head in the noose.
And then you’ll see another mechanical response because now the noose will tighten and he’s caught.
The next movie right next to it, there’s a worm coming up, I hope, should come up here.
It will stick its head in the noose and say 'no way' and it comes out.
It turns out that Mark Alkema and his lab at Massachusetts discovered
that the number of escapes that the wild type does is actually pretty good, they escape about 80% of the time.
But if you knock out their touch system that doesn’t happen.
This may be an answer of why we actually, why this vibration might actually be affecting this.
Because the animals are often found in rotting fruit, that would be in fields where there’s lots of rain.
And rain over a series of hours could actually habituate the animals to touch.
Now they are sitting ducks for these fungi, but if we have a way of reviving the systemit may actually work.
So we think this might be one of the consequences of this change.
Let me tell you another affect, now about turning it down.
Because hypoxia, dauer, formation, high salt are all conditions that worms hate.
They try to get away from them, they are trying to escape.
And we realised that under good conditions or - normally we touch the animal at the tail, it moves forward.
Touch it in the head, it moves backward, at least that’s what I told you before.
But there’s another thing that happens.
You touch in the head and it moves backwards, doesn’t go back to be touched in the same way, it veers off in another direction.
When we look in healthy animals, nice conditions, if you tap a plate - so now you are stimulating all the cells –
in fact the head overrules the tail and so the animals generally move backwards and then move off in a new direction.
But that doesn’t happen if you knock out the anterior touch system or knock it down.
So if you had under good conditions, where the animal really doesn’t care
if it gets there in time or not, not trying to get away from anything.
And every time you tap the plate, which is the tease here, the animal changes direction.
It's going to take it a while to get to the target, in this case an attractant.
However, under bad conditions, where the anterior touch system is knocked out, now mainly the tail is going to be stimulated:
every time there’s a tap it's going to move forward a little bit faster.
And so it will take a shorter period and be less distracted.
So we think these are some of the consequences of this.
I'm going to skip the next slide talking about these changes.
I want to talk about two things.
The first, there is something called Hershey Heaven, has nothing to do with chemistry neither with chocolate.
It has to do with the fact that Alfred Hershey was asked what’s your idea of 'scientific heaven'?
He said “to have one experiment that works, and keep doing it all the time.” And you learn new and different things every time.
I think of the cells that I work on as being sort of Hershey Heaven.
We have a lot of problems we are still working on, we want to know why these processes come out.
We find that the cells are engulfed by surrounding tissue.
We have really no idea why they are.
And we are looking at mutants to try to find that.
But we in fact have many other questions.
What are the downstream genes?
What organises the microtubules?
What in the world are the microtubules doing in these cells?
What determines the synapses?
What is important about that change in synapses as the animal gets older?
What other neuropeptides affect touch?
What neuropeptides do the touch cells release in their signalling?
And I call this neurohormone control, this sort-of shadow nervous system,
and we have woefully little information about that as well.
How is synaptic habituation controlled?
That’s a different topic.
And let me go to the really important one, how does touch work?
We have the products, we have no Idea how they work.
And finally, how is human touch mediated?
Are the same things happening?
All of this work is work on basic research that’s had many different consequences.
I have a nice quote by Robert Wilson that I'm going to skip because I want to spend the last 4 minutes showing you about this.
This is a little campaign I'm on, I want you to get 2 words from this slide.
I want you to get the word winner and the word FASEB, which is Federation of American Society of Experimental Biology.
They have a contest which is called 'Stand up for Science'
and they ask postdocs and students in the United States to make videos.
I'm going to show you this year's winner, last year's, 2013.
And it's wonderful and what I'd like is for this video to go viral.
So if you look up 'winner' and 'FASEB' on Youtube you will get to this video.
Some of the people in this audience will recognise some of the work described here, particularly Han and Ver.
And so let me do it: Funding for basic research.
It's a sunny day in San Francisco.
We set up a table by the water and asked people this question:
If it's the year 1960 and you have 10 Dollars to give to science,
would you spend it on a) developing an affordable treatment for diabetes or b) figuring out how bacteria protect themselves.
The majority of people we surveyed wanted to spend their money on a diabetes treatment.
But what really happened 50 years ago?
The government decided to spend money on figuring out how bacteria protect themselves against viruses.
The National Science Foundation NSF and the National Institutes of Health NIH are 2 government agencies
that allocate tax dollars to scientists.
And they decided to fund groups of researchers who thought
that studying how microbes protect themselves was an interesting question.
At the time there was no way of knowing how this basic research could affect human health.
They were simply curious about understanding something about bacteria biology.
So what did the scientists find?
Normally, when a virus enters a bacteria it adds its own DNA to the other bacteria.
In doing so the virus takes over the bacteria and uses it to make more viruses.
Scientists found that bacteria protect themselves from viruses by selectively cutting the viral DNA.
This basic finding led to a scientific revolution.
Scientists learned how to cut chunks of specific DNA and they could use this technique to move genes around.
They could even move a human DNA into bacteria.
Because bacteria are very good of making more of themselves,
scientists can use them to make large quantities of human proteins from the human gene that they introduced.
So, why was this technique useful for modern medicine?
Well, one breakthrough was the efficient production of human insulin by introducing the human insulin gene into bacteria.
This was a very significant advancement in diabetes treatment.
Before using bacteria to produce large quantities of human insulin, insulin was expensive, unsafe and less effective
because it was harvested from foetal calves.
In fact, the first person to get an injection of cow insulin suffered from a severe allergic reaction.
Because of a long line of federally funded basic research, human insulin is now an affordable treatment for diabetes.
By asking how bacteria protect themselves, the scientists discovered that bacteria can cut DNA.
This discovery was developed into a basic technique using thousands of labs around the world,
creating new knowledge and to ask new questions that led to all kinds of therapies and technologies,
including insulin, DNA fingerprinting and biofuels, the human genome,
growth hormones, cancer drugs, stroke medication, vaccines, HIV meds, antibiotics and many, many more.
Given enough time and funding, who knows what is to come?
Many other health benefits in current discoveries would have been nearly impossible
without federal funding for all kinds of basic research.
Basic research is an investment in our future.
It broadens our understanding of the world we live in and the potential benefits are limitless.
Unfortunately, science funding in the US has plateaued,
decreasing participating power and significantly hurting our competitiveness and innovation.
You may not know it but you and your tax dollars are part of the discovery process.
Thanks to you, scientists discovered that bacteria can cut DNA.
And thanks to you, insulin, other drugs and diagnostic tools have been developed and are helping patients.
So thank you for supporting basic research.
We are the scientists who depend on you.
And we are excited for the future that lies ahead.
So show this to as many people as you can.
Thank you.