Uh-oh. I’m trying to follow this talk, but it’s one for the chemistry purists: I don’t understand the words he’s saying, starting with “olefin” and continuing with “metathesis”. You’ll have to look to one of the other Lindau bloggers with more chemistry to explain it, because Schrock seems to be assuming I understand all the basics already, and I don’t.
The difference between biology and chemistry: biologists ask how natural systems and molecules work, while chemistry wants to know how to artificially re-synthesize natural biomolecules. Tsien favors a synthetic approach, asking hwo to build new molecules that will perform amusing and useful functions in biology. He compares it to architecture on a molecular scale that is also a rigorous test of one’s understanding of molecular function.
He thinks biology has the most interesting grand questions in all of science (hooray!). He also likes pretty colors. However, he thinks that interdisciplinary barriers prevent most biologists from knowing how to manipulate molecules other than nucleotides and proteins. Anyone who knows how to make non-linear molecules has an advantage.
His first target was intracellular Ca++, which we all know is central to cellular signaling. He gave a quick overview of Ca++ properties and functions, as a rationale for why he began looking at Ca++ indicators like aequorin. He started looking for something new, started with something selective for calcium, the calcium chelator EGTA, which lacks any chromophore. One way would be to add benzene rings (chromophores) to EGTA, which led to BAPTA, basically EGTA with two benzene rings. It has high calcium sensitivity and did exhibit a color shift, but a poor one. This work led to Fura-2 synthesis, in which you can still see the EGTA ancestor, but has a much more complex set of rings attached to it. Fura-2 requires microinjection, which Tsien wasn’t good at, so he synthesized a protected form of the molecule with a methyl ester protecting group that would be stripped by enzymes in the cell.
He showed a gorgeous image of the Ca++ wave in sea urchin fertilization, visualized with pseudo-colored images of calcium concentration visualized with Fura-2.
Next step: Tsien wanted to visualize cAMP, an important second messenger in cell processes. They needed a non-destructive way to assay the dynamics of cAMP, which is present at very low concentration in the presence of many other nucleotides. The idea again was to find a specific binding agent and coupling it to a chromophore. He chose to use a specific PKA subunit that binds to cAMP, and use fluorescence resonance with a pair of adjacent chromophores. It worked, and they built proteins with rhodamine and fluorescein that did the job.
They wanted a general means to fluorescently label designated proteins. This led to the discovery of Douglas Prasher’s work on cloning GFP.
What was wrong with wild-type GFP? It’s main excitation was at 395nm (UV), and only minor excitation peak at 475 (blue). We don’t like to zap cells with UV. He puzzled out which components of the molecule were responsible for particular peaks in the spectrum (which he got wrong), but by empirically juggling in different amino acids, he came up with a variant that emphasized suitable peaks in the spectrum. This work was done without the aid of knowing the 3D structure.
They worked out the crystal structure, and then with rational design, made more variants with different colors. The work was rejected by Science for amusing reasons: reviewers didn’t understand the significance. They got it published by sending a brief note to Science that announced the imminent publication of the crystal structure of wild-type GFP in Nature.
Tsien wanted to make a red version. A Russion group found a red GFP variant in a coral, which they then analyzed and found the structure. This work has led to lots of fluroescent proteins with colors from blue to red.
Problems: Sometimes FPs are too big, the excitation wavelengths are hard to get through mammalian tissues, and a few others I was too slow to get down. Darn.
They are now developing an infrared fluorescent protein based on biliverdin, and are also studying the role of proteases in cancer. They are using polycationic proteins sequences as tools to transport payload proteins into cells, with some clever tricks to regulate accessibility and make it chemically triggerable by enzymes present in tumors. They have a probe that selectively labels tumor cells, which can be a very useful guide for surgical removal of tumors. He also combines it with in vivo labeling of nerves, which surgeons don’t want to cut, making it a way to color-code living tissue for tumor resection, a technique called molecular fluorescence imaging guidance, MFIG.
This was another excellent talk — Tsien has an entertaining sense of humor.
Chalfie is interested in sensory mechanotransduction—how are mechanical deformations of cells converted into chemical and electrical signals. Examples are touch, hearing, balance, and proprioception, and (hooray!) he references development: sidedness in mammals is defined by mechanical forces in early development. He studies this problem in C. elegans, in which 6 of 302 nerve cells detect touch. It’s easy to screen for mutants in touch pathways just by tickling animals and seeing if they move away. They’ve identified various genes, in particular a protein that’s involved in transducing touch into a cellular signal.
They’ve localized where this gene is expressed. Most of these techniques involved killing, fixing, and staining the animals. He was inspired by work of Shimomura, as described by Paul Brehm that showed that Aequorin + Ca++ + GFP produces light, and got in touch with Douglas Prasher, who was cloning GFP, and got to work making a probe that would allow him to visualize the expression of interesting genes. It was a gamble — no one knew if there were additional proteins required to turn the sequence into a glowing final product…but they discovered that they could get functional product in bacteria within a month.
They published a paper describing GFP as a new marker for gene expression, which Science disliked because of the simple title, and so they had to give it a cumbersome title for the reviewers, which got changed back for publication. They had a beautiful cover photo of a glowing neuron in the living animal.
Advantages of GFP: heritable, relatively non-invasive, small and monomeric, and visible in living tissues. Roger Tsien worked to improve the protein and produce variants that fluroesced at different wavelengths. There are currently at least 30,000 papers published that use fluroescent proteins, in all kinds of organisms, from bunnies to tobacco plants.
He showed some spectacular movies from Silverman-Gavrila of dividing cells with tubulin/GFP, and another of GFP/nuclear localization signal in which nuclei glowed as they condensed after division, and then disappeared during mitosis. Sanes and Lichtman’s brainbow work was shown. Also cute: he showed the opening sequence of the Hulk movie, which is illustrated with jellyfish fluorescence (he does not think the Hulk is a legitimate example of a human transgenic.)
Finally, he returned to his mechanoreceptor work and showed the transducing cells in the worm. One of the possibilities this opened up was visual screening for new mutants: either looking for missing or morphologically aberrant cells, or even more subtle things, like tagging expression of synaptic proteins so you can visually scan for changes in synaptic function or organization.
He had a number of questions he could address: how are mechanotransducers generated, how is touch transduced, what is the role of membrane lipids, can they identify other genes important in touch, and what turns off these genes?
They traced the genes involved in turning on the mec-3 gene; the pathway, it turned out, was also expressed in other cells, but they thought they identified other genes involved in selectively regulating touch sensitivity. One curious thing: the mec genes are transcribed in other cells that aren’t sensitive, but somehow are not translated.
They are searching for other touch genes. The touch screen misses some relevant genes because they have redundant alternatives, or are pleiotropic so other phenotypes (like lethality) obscure the effect. One technique is RNAi, and they made an interesting observation. Trying about 17000 RNAis, they discovered that 600 had interesting and specific effects, 1100 were lethal, and about 15,000 had no effect at all. The majority of genes are complete mysteries to us. They’ve developed some techniques to get selective incorporation of RNAis into just neurons of C. elegans, so they’re hoping to uncover more specific neural effects. One focus is on the integrin signaling pathway in the nervous system, which they’ve knocked out and found that it demolishes touch sensitivity — a new target!
They are now using a short-lived form of GFP that shuts down quickly, so they’ve got a sharper picture of temporal patterns of gene activity.
Chalfie’s summary:
Scientific progress is cumulative.
Students and post-docs are the lab innovators.
Basic research is essential. Who would have thought working on jellyfish would lead to such powerful tools?
All life should be studied; not just model organisms.
Chalfie is an excellent speaker and combined a lot of data with an engaging presentation.
Bioluminescence is common, especially in marine organisms. Shimomura classified thes into a couple of types: luciferain, photoprotein, and an undefined “other”. Luciferin requires an enzymatic (luciferase) reaction, and luminescence is proportional to the concentration of the substrate. The photoprotein type requires a single molecule — aequorin, symplectin, pholasin, etc.
He summarized how D-Luciferin is converted to Oxyluciferin in the presence of O2 and ATP, catalyzed by luciferase, to produce light, either red light in acidic media or yellow green in alkaline media. There is also a variant of this reaction called coelenterazine-luciferase luminaescence, found in many marine organisms, like Periphylla, a ellyfish, and Chiroteuthis, a squid. Coelenterazine is converted to coelenteramide in the presence of O2 to produce light and CO2.
Another organism is Cyprodana, a crustacean that uses a luciferin variant — produces a very pretty blue glow.
Luminiscent bacteria convert luciferin into a fattye acid, FMN, to produce light, which is also luciferase-catalyzed. Luciferase seems to be popping up all over the place — unfortunately, Shimomura doesn’t talk much at all about variations in the enzyme, but is more focused on giving us the chemical intermediates produced in the reaction. Typical chemist!
This is especially unfortunate since the luciferase reaction in different organisms seems to produce very different reaction products…it’s getting me very curious about how these different forms of the protein must differ to be yielding such different outcomes, all only similar in that they also produce light as a byproduct. And then we see that some very different phyla, such as krill and dinoflagellates, use nearly identical reactions.
Photoproteins: he talks about Aequorea aequorea, which produces green light with a protein called aequorin that binds a complex molecule that resembles coelenterazine, that undergoes a conformational change in the presence of calcium ions to produce green light. If GFP is used, it produces green light.
A squid, Symplectoteuthis, converts dehydrocoelenterazine to symplectin which then produces light.
Then he switches to talking about fungal biolominescence in Mycena and Panellus, mushrooms that glow green. The precursor is decanoylpanal is conferted to luciferin, which produces light in the presence of superoxides, O2, and tetradecanoylcholine. The reaction can take place in the absence of any enzyme.
Shimomura is not exactly a dynamic speaker — he basically just read off his list of reactions — but at least he had lots of pretty pictures of glowing organisms. If only he’d said something about the relationships and evolutionary differences between them all!
This is a wonderful video: it’s Richard Dawkins interviewing Craig Venter and getting a tour of his amazing sequencing facility. It also reveals how much the technology is changing.
I am in awe — they did it without anyone noticing. They just infiltrated nations all around the planet, smuggling in individuals to form vast new colonies of billions, all loyal to the overlords back home. Of course, these are very, very short Argentinians, which made them harder to notice: they’re all ants.
In Europe, one vast colony of Argentine ants is thought to stretch for 6,000km (3,700 miles) along the Mediterranean coast, while another in the US, known as the ‘Californian large’, extends over 900km (560 miles) along the coast of California. A third huge colony exists on the west coast of Japan.
While ants are usually highly territorial, those living within each super-colony are tolerant of one another, even if they live tens or hundreds of kilometres apart. Each super-colony, however, was thought to be quite distinct.
But it now appears that billions of Argentine ants around the world all actually belong to one single global mega-colony.
You better start practicing your tango is you hope to get along with our new arthropod overlords.
This morning was a long session broken into two big chunks, and I’m afraid it was too much for me — my recent weird sleep patterns are catching up with me, which didn’t help at all in staying alert.
Robert Huber: Intracellular protein degradation and its control
This talk was a disaster. Not because it wasn’t good, because it was; lots of fine, detailed science on the regulation of proteases by various mechanisms, with a discussion of the structure and function of proteasomes, accompanied by beautiful mandalas of protein structure. No, the problem was that this listener’s jet lag has been causing some wild precession of my internal clocks, and a quarter of the way through this talk all systems were shutting down while announcing that it was the middle of the night, and I really couldn’t cope. I’m going to have to look up some of his papers when I get home, though.
Walter Kohn: An Earth Powered Predominantly by Solar and Wind Energy
Kohn has made a documentary to illustrate the power of solar energy. It was very basic, a bit silly — John Cleese narrates it — but might be useful in educating the pubic. He showed excerpts from it, and while it was nice, it didn’t fire me up.
Peter Agre: Canoeing in the Arctic, a Scientist´s Perspective
This was a bit strange. We’ve had all these science talks on global warming, so Agre decided to just show us what we stand to lose, and showed us photos of his vacations on canoeing trips in Canada and Alaska. They were gorgeous photos, but please don’t show me your photo album when I’m crashing hard.
I think my new and revised plan is to take a nap this afternoon and try to recharge a bit. I really must be alert for tomorrow’s session with Shimomura, Chalfie, and Tsien, which are the talks I was most anticipating. There’s also a curious talk by Werner Arber on something called Molecular Darwinism which has my skeptical genes tingling; I’ve got to see what kinds of evidence he provides for that. So brain must not melt down now.
I’m here for another long session of talks. Unfortunately, this is Big Chemistry day, and I’m struggling to keep up with the unfamiliar. I need more biology for it all to make sense!
Rudolph Marcus: From ‘On Water’ and enzyme caalysis to single molecules and quantum dots. Theory and experiment.
I was afraid of this. This Lindau conference has a primary focus on chemistry, and I am not a chemist…and I just knew there would be a talk or two at which I would be all at sea, and that was the case in Marcus’s talk, which was all hardcore chemistry. I got the general gist — he’s making an argument that you need both a solid grounding in theory in order to carry out computational chemistry, which seemed fairly obvious to me — but I confess that his discussion of the details of on-water catalysis, single molecule enzyme catalysis, and quantum dots lost me, through no fault of his. I don’t have the background to follow the context of the discussion.
Kurt Wüthrich: Structural genomics — exploring the protein universe
This was more of that tricky chemistry stuff, but at least it was related to biology. Wüthrich studies 3D protein structures, specializing in using NMR of proteins in solution. He fave a little background, and talked especially about his particular interest in hemoglobin, an interest that continues — he currently works at catching EPO doping in athletes. The more interesting part of the work is his current contributions to analyzing the structure of proteins in the genome. He made the point that there are currently over 6 million gene sequences tucked away in databases, but we know the the 3D structure of only about 50,000 of them. He’s part of a very large research consortium that is trying to fill in the gaps with high throughput, automated techniques.
Harold Kroto: Science, society and sustainability
If you’ve ever heard a Kroto talk, you know it is pretty much indescribable.
He did present all of chemistry in 30 seconds, but much of it was about about science education, science’s role in society, and how science is going to be necessary to save the world. There was a good strong bit of promotion of atheism (he’s one of us!), and an amusing tour of the Creation “Museum”, which he visited recently. All I can recommend is that you keep an eye on the Lindau site — they will make the lectures available online at some time.
Ah, a solid science talk. It wasn’t bad, except that it was very basic—maybe if I were a real journalist instead of a fake journalist I would have appreciated it more, but as it was, it was a nice overview of some common ideas in neuroscience, with some discussion of pretty new tools on top.
He started with a little history to outline what we know, with Ramon Y Cajal showing that the brain is made up of network of neurons (which we now know to be approxiamately 1012 neurons large). He also predicted the direction of signal propagation, and was mostly right. Each neuron sends signals outwards through an axon, and receives input from thousands of other cells on its cell body and dendrites.
Signals move between neurons mostly by synaptic transmission, or the exocytosis of transmitter-loaded vesicles induced by changes in calcium concentration. That makes calcium a very interesting ion, and makes calcium concentration an extremely important parameter affecting physiological function, so we want to know more about it. Furthermore, it’s a parameter that is in constant flux, changing second by second in the cell. So how do we see an ion in real time or near real time?
The answer is to use fluorescent indicator dyes which are sensitive to changes in calcium concentration — these molecules fluoresce at different wavelenths or absorb light at different wavelengths depending on whether they are bound or not bound to calcium, making the concentration visible as changes in either the absorbed or emitted wavelength of light. There is a small battery of fluorescent compounds — Fura-2, fluo 3, indo-1 — that allow imaging of localized increases in calcium.
There’s another problem: resolution. Where the concentration of calcium matters most is in a tiny microdomain, a thin rind of the cytoplasm near the cell membrane called the cortex, which is where vesicles are lined up, ready to be triggered to fuse with the cell membrane by calcium, leading to the expulsion of their contents to the exterior. This microdomain is tiny, only 10-50nm thick, and is below the limit of resolution of your typical light microscope. If you’re interested in the calcium concentration at one thin, tiny spot, you’ve got a problem.
Most presynaptic terminals are very small and difficult to study; they can be visualized optically, but it’s hard to do simultaneous electrophysiology. One way Neher gets around this problem is to use unusually large synapses, the calyx of Held synapse, which is part of an auditory brainstem pathway. It’s an important pathway in sound localization, and the signals must be very precise. They have a pecial structure, a cup-like synapse that envelops the post-synaptic cell body — they’re spectacularly large, so large that one can insert recording electrodes both pre- and post-synaptically, and both compartments can be loaded with indicator dyes and caged compounds.
The question being addressed is the concentration of Ca2 at the microdomain of the cytoplasmic cortex, where vesicle fusion occurs. This is below the level of resolution of the light microscope, so just imaging a calcium indicator dye won’t work — they need an alternative solution. The one they came up with was to use caged molecules, in particular a reagent call Ca-DMN.
Caged molecules are cool, with one special property: when you flash UV light of just the right wavelength at them, they fall apart into a collection of inert (you hope) photoproducts, releasing the caged molecule, which is calcium in this case. So you can load up a cell with Ca-DMN, and then with one simple signal, you can trigger it to release all of its calcium, generating a uniform concentration at whatever level you desire across the entire cell. So instead of triggering an electrical potential in the synaptic terminal and asking what concentration of calcium appears at the vesicle fusion zone, they reversed the approach, generating a uniform calcium level and then asking how much transmitter was released, measured electrophysiologically at the post-synaptic cell. When they got a calcium level that produced an electrical signal mimicking the natural degree of transmitter release, they knew they’d found the right concentration.
Caged compounds don’t have to be just calcium ions: other useful probes are caged ATP, caged glutamate (a neurotransmitter), and even caged RNA. The power of the technique is that you can use light to manipulate the chemical composition of the cell at will, and observe how it responds. These are tools that can be used to modify cell states, to characterize excretory properties, or to generate extracellular signals, all with the relatively noninvasive probe of a brief focused light flash.
Yesterday morning, I was in a discussion on UK Christian talk radio on the topic of “Is Christian faith at odds with science?”, with Denis Alexander of the Faraday Institute for Science and Religion. It’s going to be available as a podcast at sometime in the next day, but I may not be able to link to it right away — tomorrow I fly away to Germany for a week, so my schedule is going to be a bit chaotic for a while.
Don’t expect fireworks. It was the usual feeble accommodationist claptrap, but I had my nice man hat on and actually tried to get across some basic ideas. To no avail, of course, but at least I tried.
I have now discovered that I was trying to make the same points Lawrence Krauss is doing in the Wall Street Journal: religion is wrong. It’s a set of answers, and worse, a set of procedures, that don’t work. That’s the root of our argument that religion is incompatible with science.
That word, “incompatibility”, is a problem, though. The uniform response we always get when we say that is “Hey! I’m a Christian, and I’m a scientist, therefore they can’t be incompatible!” Alexander was no exception, and said basically the same thing right away. It’s an irrelevant point; it assumes that a person can’t possibly hold two incompatible ideas at once. We know that is not true. We have complicated and imperfect brains, and even the most brilliant person on earth is not going to be perfectly consistent. When we talk about incompatibility, we have to also specify what purposes are in conflict, and show that the patterns of behavior have different results.
For instance, if you just like to go to church because you enjoy the company, then the purpose of religion to you is to reinforce social bonds — so of course there is no incompatibility between science and religion there. If you go for the choir (as Stephen Jay Gould was known to do), you’re there to enjoy the music, and science does not dictate that human beings are not allowed to enjoy music. For that matter, science doesn’t say that someone is not allowed to enjoy the perverse circumlocutions of theology, so if someone attends for the religion sensu strictu, no problem.
But in a debate about the compatibility of science and religion, we have to put the argument in an appropriate context and define a specific shared purpose for both science and religion — it’s the only legitimate ground for discussion. In this case, what we’re trying to do is address big questions (remember, the Templeton Foundation says they’re all about those “big questions”) about the nature of the universe, about our history, about how we function, and then we encounter a conflict: religion keeps giving us different answers. Very different answers. They can’t all be right, and since no two religions give the same answers, but since science can generally converge on similar and consistent answers, I know which one is right. And that makes religion simply wrong.
We have to look at what they do to see why. In order to probe the nature of the universe around us, science is a process, a body of tools, that has a long history of success in giving us robust, consistent answers. We use observation, experiment, critical analysis, and repeated reevaluation and confirmation of events in the natural world. It works. We use frequent internal cross-checking of results to get an answer, and we never entirely trust our answers, so we keep pushing harder at them. We also evaluate our success by whether the end results work: it’s how we end up with lasers and microwave ovens, and antibiotics and cancer therapies.
Religion, on the other hand, uses a different body of techniques to explain the nature of the universe. It uses tradition and dogma and authority and revelation, and a detailed legalistic analysis of source texts, to dictate what the nature of reality should be. It’s always wrong, from an empirical perspective, although I do have to credit theologians with some of the most amazingly intricate logical exercises as they try to justify their conclusions. The end result of all of this kind of clever wankery, though, is that some people say the world is 6000 years old, that it was inundated with a global flood 4000 years ago, and other people say something completely different, and there is no way within the body of theology to resolve which answers are right. They have to step outside their narrow domain to get an independent confirmation — that is, they rely on science to give them the answers to the Big Questions in which they purport to have expertise.
So what theistic scientists have to do is abandon the operational techniques of religion and use science to address those questions. The “theistic” part of their moniker is nothing but useless baggage which, if they take it at all seriously, would interfere with their understanding of the world. That is what I mean by an incompatibility between the two.
Krauss uses a marvelous and well-known quote from J.B.S. Haldane to make that point more briefly.
My practice as a scientist is atheistic. That is to say, when I set up an experiment I assume that no god, angel or devil is going to interfere with its course; and this assumption has been justified by such success as I have achieved in my professional career. I should therefore be intellectually dishonest if I were not also atheistic in the affairs of the world.
I got Alexander to agree that he does not use religion in the laboratory — I don’t know anyone who would say that they do, other than creationist kooks — but it didn’t seem to sink in that that is an admission of incompatibility. Religion doesn’t work to answer questions in science, which always leaves me wondering…if you accept that, why do you go on thinking it might be giving you correct answers in ordinary daily life? It has an awfully poor track record.
Now one way the defenders of religion like to get around this empirical problem is to change the game in mid-play: one moment we’re talking about tools for understanding the world, where there is a conflict, and then they switch to a completely different purpose, that of establishing a common morality, or appreciating art, or falling in love. I would be the first to admit that science does not and should not dictate morality: the cases in the past where this has happened (eugenics comes to mind right away) have been disastrous. Science is good at explaining what is and how it works, and not so great at telling us how it should work. I also wouldn’t use the scientific method directly to determine whether I like some music or poetry or not.
However, I’m going to have to say that religion doesn’t do a good job at that either. SJ Gould tried to partition the domains of authority for science and religion by explicitly setting a boundary, and saying religion should have the job of defining what is right and good…but I think he failed, because he gave far too much credit to religion for being able to discern and act on a reasonable morality. It’s foundation on authority and its role in defining in and out groups means it is too exclusionary, too narrow and inflexible, and also too willing to ignore empirical evidence. It’s why we have religion behind such immoral acts today as trying to restrict civil rights to people who have only a certain range of sexual behaviors, or facilitating the spread of sexually transmitted disease in Africa by damning sex education and condom use.
And when it comes to other questions than the cosmic ones about the nature of existence, I prefer that we apply just about any discipline other than religion to the problem: at least they are evidence-based, where religion is not. I’d rather consult a philosopher than a theologian on morality; they’ve been thinking about it with a broader scope than the pious promoters of sectarian belief, anyway, don’t restrict their principles to worshippers of one particular idol, and usually don’t invoke magical rewards and punishments that have never been seen to justify decisions. If I’m in love I’m better off pulling a book of poetry off the shelf than consulting a celibate. I’d rather hear about economics from an economist than from a ouija board or a pulpit, and I like the idea of policy decisions being evaluated for effectiveness, rather than ideological purity. When we’re looking at communities and interactions between individuals, give me a psychologist or a sociologist over a priest any day. The only useful priests in those matters are the ones who understand the principles of psychology and sociology, and apply those, rather than pulling a quote out of their holy book.
Accommodationists are a problem not because accommodation is bad, but because they are pushing for the wrong kind of accommodation. Science doesn’t need to conform, religion does. Religion demands a special kind of privilege in these discussions because if we actually get down to assessing views fairly and objectively, on the basis of what works, it fails. I say, let it.
This is also why so many of us object to the Templeton Foundation. Their agenda consists solely of mixing up science and religion, to the detriment of the former. They just want to compromise…but asking us to compromise science that works with faith that doesn’t is a fool’s bargain. Why should we?
