Volume 1, Issue 7
Reengineering Cells To See
Dirk Trauner has a grand vision. The UC Berkeley chemist hopes that the success he and his colleagues have had making blind cells "see" could someday lead to a cure for human blindness.
Before joining the UC Berkeley faculty, Dirk Trauner was a postdoctoral fellow at the Sloan-Kettering Institute for Cancer Research.
"We've borrowed one of nature's molecular machines, reengineered it, and put it back into a cell to add new functionality," he says. "If you could re-educate a neuron to be light sensitive, you might be able to create an artificial retina."
Trauner and Richard H. Kramer, a professor of molecular and cell biology, are taking the first steps toward that futuristic goal. The researchers have already inserted a biological switch into a rat brain cell so that it can be turned on or off in response to different frequencies of light.
Their technique, Trauner explains, could eventually become the basis of a gene therapy for macular degeneration, the leading cause of legal blindness in people over 55. Macular degeneration refers to the degradation and death of the nerve cells in the retina that collect the light that the brain uses to construct a picture of the world. In macular degeneration and other similar diseases, the nerve cells downstream from the photoreceptors are still alive though, making them an interesting site for a possible treatment.
"Many of the living neurons that project the signals into your brain also turn out to be on the surface of the eye," Trauner says. "That means they can be easily reached by painlessly injecting the eye with a harmless virus that would carry the therapeutic gene into the correct cells."
The idea behind the gene therapy is that by tricking cells into responding to light, they would fill in for the photoreceptors knocked out by disease. The researchers' targets are the tiny electrical gates in a cell's membrane that govern such basic function as signaling, regulation, and excitability.
Working with molecular and cellular biology professor Ehud Isacoff, an expert in the dynamics of ion channels, the researchers focused on potassium channels. These channels are proteins that function like pores, selectively letting potassium pass in and out of the cell. The channels are triggered by changes in voltage in a mechanism familiar to electrical engineers.
Potassium channels normally open in response to a voltage difference between the inside and outside of a nerve cell, letting potassium ions (K+) flow out to equalize the voltage and turn the cell off. This channel has been broken, then re-engineered to open when hit with ultraviolet light and close when hit with green light. The opening and closing is achieved with a molecule that kinks and unkinks in response to different wavelengths of light. This photoswitch can be used to selectively silence nerve cells or to give the gift of "sight" to normally sightless organisms or cells. (courtesy the researchers)
"A voltage-gated ion channel is nature's version of a field-effect transistor," Trauner says. "It's a device that changes its conductance based on the voltage applied. However, we wanted to make its conductance change in response to light."
To add the new functionality, the researchers mutated a gene for the ion channel so that it remains open and added an extra molecule that would act as a hook for a photosensitive molecule. They then introduced the mutated gene into cells from a rat's hippocampus, a sliver of the rodent's brain. Next, they washed the hippocampus cells with a photosensitive chemical, called an azobenzene compound. The azobenzene molecules bind to the hook on the potassium channel and function like a stopper in a sink drain. Depending on the wavelength of light hitting it, the stopper either plugs the pore or is yanked out of it.
A computer-generated visualization of a potassium ion channel structure. (courtesy the researchers)
Based on the promising in vitro results with the rat hippocampus, Trauner and his colleagues will next apply the technique to mice that they've genetically engineered for blindness comparable to the kind caused by macular degeneration.
"We'd like to demonstrate that the blind mice can become sensitive to light," he says. 'We're not even dreaming yet of color vision or real spatial resolution."
In the shorter term, the light-sensitive ion channels could also be used in more basic research on the brain. Scientists might use lasers to selectively activate or silence certain neurons to gain insight into the precise function of various neuronal circuits.
The development of synthetic ion channels could have other applications as well. For example, an ion channel might be synthesized that's sensitive to heavy metals or various pathogens in the air. The channel could then be used as an ultra-sensitive environmental sensor.
"We're taking a basic building block of life and souping it up to do something it did not evolve to do," Trauner says. "This is true synthetic biology."
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A Flare Up In Solar Physics
The sun is the solar system's biggest powder keg. Solar flares, tremendous explosions on the sun's surface, can release the equivalent energy of 10 billion megatons of TNT in just a few minutes. The explosions sometimes interfere with terrestrial radio communications and might put astronauts' lives in jeopardy, but the complex phenomena that ignite the flares remain much of a mystery. Six hundred kilometers above the earth though, a small satellite operated by UC Berkeley scientists is shedding some light on the sun's unrest.
Scientists believe that solar flares occur when energy stored in the magnetic fields of the sun's corona is suddenly released. Electrons and ions are explosively flung toward the surface of the sun, heating the solar atmosphere to tens of millions degrees Celsius. Sometimes, up to a billion tons of gas is ejected into space. In recent years, scientists determined that perhaps as much as half of the energy released by the flares is contained in accelerated electrons and ions.
Robert Lin at the Space Sciences Laboratory. Visible in the background is the eleven meter-diameter dish in the Berkeley hills used to communicate with the RHESSI satellite. (David Pescovitz photo)
"Solar flares are the most energetic particle accelerators in the solar system," says UC Berkeley astrophysicist Robert Lin, director of the Space Sciences Laboratory. "But how the sun releases the energy and accelerates the particles with such high efficiency is a puzzle that couldn't be solved on the ground."
Three years ago, NASA and UC Berkeley launched the Reuven Ramaty High-Energy Solar Spectroscopic Imager (RHESSI), a satellite designed at the University to observe the energy released by the flares before it's absorbed in the Earth's atmosphere. RHESSI is outfitted with novel imagers that measure x-ray and gamma rays produced when electrons and ions collide with the solar atmosphere. Those rays, Lin says, provide the most direct signatures of the location and motion of the particles released by the flares. The trick is imaging them.
The rays are of such high energy, Lin explains, that they cannot be focused by the lenses or mirrors of traditional optical telescopes. To solve that problem, Lin's research group developed the RHESSI Imaging Spectrometer based on grids that block some photons while allowing others to fly through the gaps. Photon detectors behind the grids not only count the particles passing through, but also precisely measure the energy of those photons.
As the satellite flies over UC Berkeley, those measurements are transmitted to an eleven-meter diameter dish on the hillside outside the Space Sciences Laboratory. The data is then combined to construct high-resolution spectrographic movies of the flares.
A superposition of RHESSI images of gamma-ray and X-ray emissions with a TRACE satellite extreme ultraviolet image taken 90 minutes later of the July 23, 2002, solar flare. The superposition clearly shows the large separation between the high-energy emissions. Solar physicists expected to see X-rays and gamma rays emerging from the same spots at the base of the flare loops.
"When we image the acceleration of the particles, we can see where the energy is coming from relative to the sun's magnetic field," Lin says. "And the spectroscopy enables us to measure the conditions in that region such as density and temperature. Once we know where the activity occurs and the conditions surrounding it, we can then begin to understand the physics behind the flares."
Already, RHESSI has enabled Lin's team to produce dazzling movies of the unusual phenomenon with unprecedented clarity. Towards the end of October 2004, the satellite measured some of the biggest flares that the sun may have ever unleashed. "It's these big flares that really push the limits of our understanding," Lin says.
Solar flares are produced by the sun's massive magnetic fields. Those fields are commonly seen as sunspots, "active regions" where the solar magnetic field may be several thousand times that of the Earth. As those magnetic fields twist like tightly-coiled springs, the tension eventually becomes too much and they snap, spewing the charged particles that make up a solar flare.
The RHESSI Imaging Spectrometer contains nine germanium detectors that are positioned behind the nine grid pairs on the telescope. The detectors convert incoming x-rays and gamma-rays to pulses of electric current. The amount of current is proportional to the energy of the photon, and is measured by sensitive electronics designed at the Lawrence Berkeley National Laboratory and the Space Sciences Lab., Berkeley. (NASA photo)
Before last year's observations, solar physicists expected that the accelerated ions and electrons would be seen along the same magnetic pathways. Both gamma rays and x-rays, the scientists believed, should originate from the feet of massive loops that arch hundreds of thousands of miles across the surface of the star. However, once RHESSI's data made it home, the scientists were pleasantly surprised.
"What we found is that the electrons and ions were separated by tens of thousands of kilometers," Lin says. "We don't completely understand why that's the case, but it suggests that the particles are accelerated in different ways."
The massive amounts of data already delivered by the satellite will take years to fully analyze, he adds. As long as funding continues, Lin says, RHESSI's spectroscopic eye will remain open for the foreseeable future.
"If you truly understand what causes the flares, someday you might be able to look for the right conditions and predict when the flares will occur," he says.
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Fly Guy
Nestled inside the human genome, there may be another secret code waiting to be deciphered. The human genome is now thought to contain 22,000 or so genes that code for proteins, the building blocks of life. But how are such a small number of genes programmed to embark on widely different paths of development? In other words, says UC Berkeley molecular and cell biologist Michael Levine, "what puts your head on top of your shoulders and not in your rear end?" To answer that question, Levine has spent several decades reigning over his genetics laboratory as lord of the flies.
Michael Levine is a professor of Genetics and Development and is also affiliated with the Division of Cell and Developmental Biology.
"Flies may be small, but they're up there with vertebrates in terms of complexity," Levine says. "I'm a fly guy for life."
Indeed, Drosophila, the common fruit fly, is ideally-suited for Levine's experiments that provide insight into the mind-boggling intricacies of fetal development. The researchers focus on the second hour of a fly's embryonic life. That's when the cells begin to differentiate into various parts of the fly tissue and the signs of regulatory DNA are first revealed. Regulatory DNA, Levine explains, controls how and where a gene is expressed in a cell. Of the three types of regulatory DNA--enhancer, silencer, and insulator--"enhancers are king, activating gene expression in specific cell types for specific tissues," he says. Scientists conservatively estimate that while the human genome has less than 30,000 genes, it may contain 100,000 enhancers at the minimum. So far, just 50 or so have been identified.
This confocal microscopy image shows a potential enhancer-promoter loop. The two copies of the gene are closely linked. Red identifies the promoter, blue the proximal (or nearby enhancer), and green the distal (or remote) enhancer. The copy of the gene on the right shows the expected linear order: promoter, nearby enhancer, distant enhancer. However, the gene copy on the left shows the remote enhancer (green) near the promoter (red), with the nearby enhancer (blue) displaced away from the promoter. The simplest interpretation of this structure is that the distant enhancer has formed a looped structure to the promoter. (courtesy the researchers)
"It's hard to come up with an accurate estimate because they're so elusive," Levine says. "You can take an unknown genome and find a protein-coding gene just by reading the code. You may not know a thing about the gene, but at least you can identify it. So far though, we haven't found the code for regulatory DNA, if one even exists."
In recent years, Levine has leveraged computational methods to sift through genetic data in search of binding sites within certain genes that may be indicative of a "landing pad" for enhancers. As his research group continues to build a dataset of possible enhancers, they've also begun to examine enhancers function. Specifically, how does an enhancer activate a gene when it maps thousands of base pairs of DNA away?
For several years, scientists have believed that the DNA between the two genes loops outward, enabling the enhancer to physically land near the appropriate promoter, the part of the gene that kicks off the protein synthesis process by triggering the transcription of mRNA. Still, the evidence of this flopping mechanism behind long-range enhancer-promoter communication was indirect. Scientists could see the promoter region of a gene, but not the enhancer. Until recently.
In this confocal microscopy image, the two copies of the gene are closely linked. The copy on top displays an extended organization of the promoter (red), nearby enhancer (blue), and distant enhancer (green). The copy below shows a compact organization. Note that the distant green enhancer (green) on the compact chromosome is in very close proximity with the promoter (red) on the extended chromosome. This might represent a direct visualization of transvection. (courtesy the researchers)
Levine and postdoctoral researcher Matt Ronshaugen devised a way to visualize the remote enhancer. The researchers' trick was to leverage another known but mysterious phenomena in the genome. The enhancers themselves are coated with RNA. Nobody understands why, Levine says. The RNA could be an unimportant byproduct of a nearby promoter or it could help recruit proteins that are essential in the looping process. In any case, the non-coding RNA enabled the researchers to apply a time-tested technique of visualizing genetic material called RNA-FISH (RNA-Fluorescent In Situ Hybridization). Using RNA-FISH, the researchers tagged the RNA associated with the enhancers with a green fluorescent molecule. Then, using a confocal microscope, they literally watched the green-tagged enhancer loop over to the red-tagged promoter gene.
A serendipitous surprised followed. In 1954, Nobel Laureate Ed Lewis, whom Levine calls the "Einstein of flies," proposed that a gene on one chromosome can directly affect the expression of its homologue gene on another chromosome, a process called transvection. However, the frequency of this "crisscross" was unclear. Levine and Ronshaugen observed that, at least in the case of fruit flies, transvection is quite common.
"One possible explanation for transvection is maybe that it's used as a homeostasis mechanism," Levine says. "If an enhancer fails on one chromosome, the other chromosome can compensate. That way you make sure to get the right levels of expression."
Understanding the myriad molecular processes of fetal development, Levine says, could someday help scientists realize the promise of the post-genome era.
"Could we determine that a certain gene will get turned on in the prostate of a 50-year-old man just by reading off the sequence?" he asks. "It's possible, but we're nowhere near that. The fly embryos are a simple model that helps us determine if there's any underlying code that we can crack."
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Berkeley's Scientific Legacy
1940: Edwin M. McMillan and the transuranium triumph
For many years, scientists believed that Uranium, with its atomic weight of 92, was the upper limit of the periodic table. But in 1940, more than a century and a half after Uranium was first discovered, UC Berkeley physicist Edwin M. McMillan, working with Philip Abelson at Lawrence Radiation Laboratory, boosted the number of known elements to 93. Neptunium led the way for the discovery of many other elements heavier than Uranium, and the development of various nuclear fuels.
Nobel Laureate Edwin M. McMillan
During those early days at the Lawrence Radiation Laboratory, Ernest O. Lawrence ran a tight ship with a limited budget. "You had to be a theorist, experimentalist, and an engineer," McMillan once said. Still, Lawrence's newly-invented cyclotron enabled the researchers to conduct groundbreaking experiments with huge scientific pay-offs.
In some ways, McMillan and Abelson succeeded where famed Italian physicist Enrico Fermi fell short. In 1934, Fermi claimed that by bombarding uranium with neutrons, he had converted it into a new element, number 93 on the periodic table. It was later found that Fermi had actually split the uranium atom, demonstrating nuclear fission, but not another element.
With the aid of the cyclotron though, McMillan and Abelson conducted their own fission experiments and eventually produced a true sample of element 93. Following the naming of uranium, the new element was dubbed neptunium for the next planet out in our solar system.
Edwin McMillan the year he discovered neptunium
McMillan and his collaborators went on to find early evidence of element 94. However it was during those experiments that he was called to MIT to conduct research for the war effort. There, he helped develop radar and sonar and, later at Los Alamos, worked on the atomic bomb. Back at the Berkeley Lab, Glenn Seaborg and his colleagues continued McMillan's experiments and eventually confirmed the discovery of plutonium. That line of research made headlines when the atomic bomb was dropped on Nagasaki, ending the war.
In 1951, McMillan and Seaborg shared the Nobel Prize in Chemistry for their "discoveries in the chemistry of transuranium elements."
After the death of Lawrence in 1958, McMillan was appointed director of the Lawrence Radiation Laboratory at Berkeley and Livermore. Shortly after, he discovered "phase stability," a principle that massively increased the energy of particle accelerators. The findings led to McMillan's invention of the synchrotron, still a key instrument in nuclear physics research. He and Russian physicist VI Veksler shared a 1963 Atoms for Peace Award for their independent work in this area.
In 1971, the Lawrence Berkeley Laboratory and the Lawrence Livermore Laboratory split into two entities as they exist today. McMillan directed the former until his retirement in 1973. He died in 1991.