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!
It’s another exciting day of exciting lectures, I hope. I know that this morning was the most anticipated one on my dance card; here’s what we’re looking forward to.
Osamu Shimomura: Chemistry of Bioluminescence
Martin Chalfie: GFP and After
Roger Y. Tsien: Building and Breeding Molecules to Spy on Cells, Tumors, and Organisms
Richard Royce Schrock: Recent Advances in Olefin Metathesis Catalyzed by Molybdenum and Tungsten Alkylidene Complexes
Werner Arber: Molecular Darwinism
OK, I confess, Schrock’s lecture won’t be my cup of tea, and I have no idea what his title says, but the rest sound fun, and the last one sounds controversial. I’ll be back in a few hours with some short summaries.
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 received Chris Mooney’s last two books as review copies, before the simple folk could get theirs, and I also gave them positive (and sincere!) reviews. I’d noticed that he’s got a new book out, but strangely, I hadn’t been sent a copy this time. I was wondering what was up with that, but now Ophelia Benson has read part of the book, and all is explained. He spends part of one chapter singling me out for criticism! Gosh, I guess he felt he wouldn’t get a friendly review this time.
The focus of his ire? Crackergate. He regards destroying a sacred symbol to be inflammatory and obnoxious, completely ignoring the insanity it exposed. That insanity — and I am not using that word casually — is what Mooney thinks the spokespeople for science in our country ought to treat deferentially. Here’s why he thinks we need to do that:
America is a very religious nation, and if forced to choose between faith and science, vast numbers of Americans will select the former. The New Atheists err in insisting that such a choice needs to be made. Atheism is not the logically inevitable outcome of scientific reasoning…A great many scientists believe in God with no sense of internal contradiction…[pp 97-98]
Ah, yes, the policy of cowardice. We are weak, and the loons are numerous and strong, and therefore we must avoid telling them the truth. If the masses prefer their silly religion to science, well then, we shall give them a neutered science, a weak science, an inoffensive science that does not challenge anyone’s beliefs.
If atheism is not the logical outcome of scientific reasoning, then let us pretend that gods that turn into a cracker that cures you of sin is logical and unquestionable and harmless…oh, wait, let’s pretend that belief doesn’t exist, and doesn’t poison minds. We’ll blame the American problem of unreason on the atheists, instead.
Sheril Kirshenbaum assures me that I will be receiving a review copy of their book; I’m not being intentionally snubbed, it is merely a matter of timing, and the review copies are only now being sent out. I look forward to it with grim anticipation. I am hoping that the rest of the book isn’t as awful as chapter 8, or I’ll have to be brutal.
Do you recall this incoherent tale of incest and racism I got a while back? Like almost 4 months ago? Well, the fellow seems to have just now discovered that we were laughing at him, and has sent me his rebuttal. It’s only fair that I post it for you now.
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.
