First Cell

Part of the definition of life, says David Deamer, is that it is in a place.  Deamer is not uttering a koan in a Zen monastery. He’s sitting next to a microscope in a biology laboratory at the University of California at Santa Cruz. Deamer is a hard-core biophysicist, but still there is a monkish quality to him.  It comes not just from his unnervingly gentle manner of speaking but from his entire approach to science. This is a man who, in contemplating the pattern of nucleotides in DNA--represented by the letters A, C, G, and T--was reminded of musical notation. By allowing the letters to stand for notes instead of nucleotides--and using E as the equivalent of T--he turned human DNA into hypnotic melodies, available now for your meditative pleasure on both tape and CD. Deamer himself likes to hum the insulin gene. This is a man who isolates chemicals from meteorites and asks guests in his lab, Do you want to smell outer space?  It doesn’t hurt to have such a cosmic view of things in Deamer’s chosen field of study: the origin of life. Deamer is unusual even among the few dozen researchers in his field, and not just in his discography. For most of the others, explaining the origin of life means explaining the origin of the genetic code: How did DNA arise from chemical reactions on the early Earth? How did the original building blocks of today’s genetic code assemble themselves into crudely self-reproducing units? Were the first life-forms based not on double-stranded DNA but on single-stranded RNA?  For the past 18 years, though, Deamer has been gently reminding his colleagues that these questions define only part of the puzzle of life. DNA does not float loosely through the oceans. Life is constrained in a place--or, to be more specific, within a boundary. Life is chemical interaction, and for that interaction to occur, life’s molecules must be close to one another. Without a physical boundary of some sort, without a skin, a bark, or a cell membrane, an organism is nothing more than a diffusing blur of molecules. To explain how the first creature came to be, you have to explain how its innards got to be distinguished from its surroundings. In other words, you’ve got to explain how the first single- celled creature got encapsulated in a cell.  Over the years Deamer has persistently been teasing out some answers to this thorny question. Now he has reached a milestone. Under conditions something like those on the early Earth, he can create something like a cell: an enzyme-carrying bubble that draws in nutrients from its surroundings and crafts them into genetic material. Call it a quasi cell-- and say that Deamer has created quasi life.  A cell membrane’s importance to life is often underappreciated, says Deamer. People say, ‘Well, it’s just a little bag.’ But it’s much more. It’s the interface between life and everything that’s outside. The membrane of any cell has to do many things at once. It has to be impermeable enough to keep essential things (like DNA) in and harmful things (like viruses and poisons) out. Yet a cell membrane can’t form a perfect seal. It has to be able to flush out waste and heat from its own system and take in nutrients from the surrounding medium. And the first cell membrane, like the membranes of many single-celled organisms today, probably had to be able to collect energy as well.  When Deamer began his work on membranes as a graduate student in the early sixties, biologists were just learning what membranes were made of: thin films of oil composed of molecules called lipids, tadpolelike things with little heads and long tails. The heads are made of charged groups of atoms, such as sugars or phosphates, while the tails are long chains of uncharged carbon and hydrogen atoms.  All cells exist in a watery world. A water molecule, though neutral, can behave as if charged because of its polar structure: the oxygen atom pulls the electrons of the two hydrogen atoms toward it, making the oxygen end of the molecule more negative and the hydrogen more positive. This polar structure is the basis for an interesting relationship between water molecules and lipids: the lipid’s charged head can form a weak bond with a water molecule, but the uncharged tail cannot. Thus, in a cell wall, lipids are usually arranged in sheets made of two layers, with the lipids in each layer pointing in opposite directions. The water-loving heads contact water both inside and outside the cell, while the water- loathing tails stay tucked safely within the wall’s oily interior.  Arranged this way, lipids make surprisingly good barriers. A neutral or a weakly polar molecule, like water, can pass through without much effort--thus ensuring that cells won’t dry out. But a fully charged molecule, or ion, trying to fight its way through the uncharged lipid tails needs a lot of energy--10,000 times more energy than that needed by a water molecule. That’s why if you try to dissolve salt in oil, it’s not going to work; it’s just going to sit there, says Deamer. Thanks to lipids, therefore, a cell can keep out harmful ions while holding in ions it uses in the production of energy.  In the early sixties biophysicist Alec Bangham of the Animal Physiology Institute in Cambridge, England, made a remarkable discovery about lipids: they can put themselves together. When he extracted lipids from egg yolks and threw them into water, he found that the lipids would naturally organize themselves into double-layered bubbles roughly the size of a cell. Bangham’s bubbles soon became known as liposomes.  Deamer was intrigued when he learned of these cellular shells. In 1975 he went to England to work with Bangham, taking a sabbatical from the University of California at Davis, where he then taught. Together Bangham and Deamer thoroughly studied the self-created liposomes, figuring out ways to increase their volume. That work helped open the door to subsequent research that has made it possible to use liposomes to carry drugs and repaired genes into the body. And those discoveries have attracted an intensely interested audience of biotechnology firms.  But it was then, 20 years ago, that Deamer and Bangham also realized that liposomes might have provided life’s first shelter. Previously, researchers had assumed that membranes were always built the way they are now--by an intricate system in which DNA provides the blueprints for the structure, and RNA ferries the instructions to the cell’s protein-making factories. If so, then genetic molecules had to exist before membranes. But studies of liposomes demonstrated that if lipids existed at the dawn of life, they would naturally, and quickly, have formed simple, albeit empty, membranes.  If there were lipidlike molecules on the early Earth, says Deamer, there must have been membranes that would have predated life. They would have been just hanging around there as little bubbles until something came along to inhabit them. These bubbles might have engulfed early molecules that had the crude ability to replicate. The liposomes would thus be able to protect them from their harsh surroundings and concentrate them so that they could react (and evolve) quickly and efficiently.  When he returned to Davis, Deamer pursued the membrane first hypothesis, experimenting with mixtures of three compounds researchers believed existed on the early Earth: fatty acids, glycerol, and phosphates. In the right concentrations, he found, they formed into lipids, and in turn, the lipids spontaneously assembled into liposomes. Now Bangham’s ponderings had turned into some real chemistry, and Deamer’s journey to life’s genesis had begun.  The waves that crash on the shores around Santa Cruz must first travel over the jungles of kelp in Monterey Bay. They pick up some of the flotsam of the underwater forests: loose seaweed, the rare seal’s corpse, fragments of countless dead plant and animal cells. As the waves come closer to land, the lipids in this cellular debris rise to the surface and lift their water-hating tails to the air. The waves mix them together and they join into bubbles. This cream-colored foam is different from the normal silver froth of churning water; its bubbles are so stable that it holds together on the water’s surface. Sometimes when the waves reach the coast, the foam shoots through channels of rock like eggnog blasted from a fire hose. Other times it collects offshore into long ribbons, then rides up onto the sand and into tide pools, where it sits quivering in the wind.  A short trek inland, in a grove of redwoods, is Deamer’s new lab, where he has been for the past year. Santa Cruz is a more appropriate setting for his work than the flat farms around Davis; what is happening down on the beach is much like what Deamer thinks happened at the dawn of life.  To demonstrate those first steps, Deamer repeats an experiment he first did a dozen years ago. At the time, he explains, many researchers resisted the membrane-first hypothesis precisely because a liposome is so impermeable. A conceptual barrier that everyone then had in their head was that there couldn’t have been membranes on the first forms of life because you couldn’t get big molecules inside them. But there’s actually an easy way, he explains as he picks through a frosty tray of assorted vials he has taken out of the laboratory freezer.  He opens a jar of lipids, extracted from egg yolk, and mixes some of the clear oil into a small test tube of water. To the naked eye the water seems unchanged, except that it has taken on a slightly milky quality; in actuality it is now full of microscopic bilayered bubbles. Deamer extracts a few drops from the mixture and puts them on a glass slide. With the casual precision of a veteran chef, he then adds dried white threads of DNA from salmon sperm to a second test tube, where they turn gooey. He spikes the solution with a fluorescent stain and adds some of these DNA drops to the lipids on the slide. Why don’t we get the hot plate going? he says to Ajoy Chakrabarti, his postdoctoral researcher. Chakrabarti switches it on and puts the slides on its surface.  That’s our tide pool, Deamer says, nodding toward the hot plate. Imagine a primitive sun beaming down on that. We’re going to let it dry down. The bubbles are moving around, and pretty soon, as the water leaves, they touch. They fuse and you have enormous planes of lipids. If anything is in between, it gets sandwiched between the planes.  After a few minutes of primordial heat, the lipids and DNA on the slide have dried into a thin film. Deamer fills his tide pool again by adding a few drops of water. He puts it under a fluorescent microscope, and Chakrabarti turns out the lights. Looking through the eyepieces, you can see lipids squirting out from the dried film into the surrounding water. At first they writhe like snakes; gradually they swell into bubbles. Some of them are dim, but others glow with the intense fluorescent green dye attached to the DNA. The glow is clear proof that as the planes of lipids curled up into vesicles, the DNA that had been sandwiched in between them got trapped inside.  There are many exotic new ideas these days about where life originated. Some researchers say the grand event took place around the furnaces of underwater hydrothermal vents; others look in the spray of ocean bubbles; and still others prefer clay. But Deamer’s choice is tide pools, an idea that harks back at least as far as Darwin’s warm, still ponds. Twenty years ago researchers showed that the wet and dry cycles of actual tide pools could bond together several precursors of RNA. It seemed reasonable to think that these pools could have been the cradle for genetic molecules, and it was likely that liposomes would have sloshed into the pools as well. All this organic stuff is accumulating on early beaches, Deamer says, and the sun is heating and drying it, and lots of natural experiments are taking place that I’m trying to re-create in the laboratory.  But a decade ago Deamer began to have doubts about the materials he was using. Astronomers and geologists were discovering that Earth had a violent infancy--hundreds of millions of years after the planet had formed, giant asteroids and comets still crashed into it, burning off its young atmosphere and boiling away its oceans. In the process, they also destroyed all the chemicals that researchers assumed were in liberal supply on the early Earth, including the building blocks of lipids. There were some naive aspects, but I was playing by the rules of the time, Deamer admits of his early research. Still, given that there must have been a first cell, it had to have a source of lipid molecules. It had to.  Research now suggests that the source was extraterrestrial. Comets and meteorites evidently brought seeds of creation to replace the ones they had destroyed, in the form of hundreds of different organic carbon molecules synthesized when the solar system was a swirling disk of gas and dust. After the last atmosphere-killing impacts--about 4 billion years ago--smaller comets, meteorites, and dust from space could, in the space of a few hundred million years, have brought enough organic carbon to cover the planet in a layer ten inches deep.  Deamer wondered whether space could also supply him with his membranes; specifically, he wondered whether he could dig them out of a 200-pound meteorite that had fallen in Murchison, Australia, in 1969 and that was positively tarry with organic carbon. In 1985 he traveled to Australian National University in Canberra to study it. The question was, he says, are there any things in the meteor that form bilayers? If so, it would be fair to assume that after impacts of similar meteorites in the ocean billions of years ago, such substances could have washed up onshore in a tide pool, dried, and then been rehydrated.  Deamer ground a piece of the Murchison meteorite and extracted the organic carbon, made it into a slurry, dried it, and then added water again. I took that ordinary extract and put it on a slide; I didn’t know what I was going to see. It was a wonderful surprise--the whole slide began to fill with these beautiful little vesicles. I started taking pictures immediately. It’s like what they say about seeing a UFO--you want to get your shots in. I can remember running downstairs to a lunch group of my colleagues and showing the pictures, and they looked at them and said, ‘From meteorites?’ It was pretty hard to believe.  Since then Deamer and his co-workers have tried to figure out exactly which of the meteorite’s molecules form these membranes. We found a few things we can identify. The problem is that meteorites are such complicated things with hundreds of chemicals, and we’re stuck with just a few precious micrograms to analyze. One substance they have isolated is nonanoic acid, a chain of nine carbons, and they’ve managed to form membranes with it. Yet their membranes fall apart sooner than the ones formed from Deamer’s original stew, which suggests that the true membrane formers are probably still hidden.  Incidentally, it was during this work that Deamer found the aroma of outer space--it smells like a musty attic. Think about it, he says after some observers put their nose to the vial of meteoritic organic carbon. You now have molecules in you older than the Earth. Deamer jokes about marketing it as a cologne--Chanel Number Five Times Ten to the Ninth, he’d call it.  The scent of meteorites, though, might put researchers on the trail toward discovering how the first organisms harnessed energy. The musty odor comes chiefly from a group of chemicals named polycyclic aromatic hydrocarbons, or PAHs for short, that are made of hexagons of carbon and hydrogen atoms linked in various arrangements. PAHs are unpleasant stuff--you can find them coming out of almost any tailpipe--but they may have made life possible on early Earth. Some years ago researchers discovered that when a PAH is exposed to light it can give off an electron. That’s what chlorophyll does for plants, explains Deamer. Plants capture the energy of this free electron and use it to bind together carbohydrates. It’s possible, Deamer thinks, that in a similar manner PAHs could have supplied energy to early cells. He has managed to incorporate PAHs into lipid membranes. Now, he says, we’d like to make them capture energy in a useful form. Nobody’s particularly impressed yet, but we think we may be able to capture carbon dioxide and use the light energy to attach it to something else.  Deamer was encouraged by this work--he had found hints that meteorites supplied material to form membranes that could have enclosed complex genetic molecules and could have trapped energy. But how do you get from there to a cell? One big problem was that these early membranes would simply have been too good at separating what they enclosed from the environment outside. A cell needs to pull in ions and toss them out all the time, so it overcomes its membrane’s impermeability with intricate channels, pumps, and shuttles. Swallowed by a liposome, a primitive genetic molecule would have been unequipped to manufacture channels through the membrane. The liposome would not be a shelter but a prison--or at least, so it seemed.  People think that membranes are permeable to nutrients and ions only if you put a channel through them, says Deamer. That’s the end of the story, because that’s the way it’s brought up in textbooks. But he has recently discovered that the textbooks are wrong.  Modern cells contain lipids with tails 16 to 18 carbon atoms long, with the rare 14-carbon tail appearing in some microbes. Tails with 12 or fewer atoms don’t appear in any cell membranes, anywhere. To determine the effect of tail length on permeability, Deamer prepared lipids with a range of tails and tried to make liposomes with them. By measuring how well they could trap charged dye molecules, he could measure their impermeability. Short tails, he found, couldn’t form bilayers at all; the best they could manage were little clumps of particles. Lipids with tails of at least 16 atoms, on the other hand, formed tightly sealed liposomes that held their dye stubbornly. However, tails with 10 to 14 atoms could also form liposomes, though they were leaky. The tails evidently weren’t quite long enough to form a permanently stable barrier, and occasionally some of them would jiggle around and create a pore. No longer is it a pure oil across there, Deamer explains. You’ve got a defect that creates a space through which ions can leak. The defect might last for only a millisecond, but you have billions of ions striking a membrane per second, and if something opens even for a microsecond, maybe ten ions will squirt through.  In 1990, Deamer started trying to toss ions through these pores. Potassium ions, he found, would go through nicely. In 1992, Chakrabarti managed to slip amino acids, which are three times bigger than potassium, through the leaky membrane. Perhaps, the researchers speculated, the earliest membranes were made of such short-tailed lipids; then, once the first cells had the genetic machinery up and running to make protein channels, they could make lipids with longer tails for better insulation without starving themselves. But Deamer and Chakrabarti still faced an intimidating challenge. For their hypothetical scenario to work, they would have to show that truly significant biochemistry could happen inside their liposomes. And to achieve that kind of chemistry, they would have to provide an encapsulated enzyme with a steady diet of much bigger genetic molecules. If the potassium was the size of a walnut, these molecules might be the size of a watermelon, explains Deamer.  At this point, Deamer and Chakrabarti teamed up with molecular biologists Gerald Joyce and Ron Breaker, at the Scripps Research Institute in La Jolla, California, who have made the study of an RNA-based artificial life something of a specialty. The researchers began by forming liposomes out of 14-carbon lipids and used Deamer’s tide pool method to capture an enzyme known as an RNA polymerase. In modern cells this enzyme grabs nucleotides and puts them together into RNA. Four nucleotides are needed to make real RNA, but for simplicity’s sake, Deamer and his co-workers used only one.  They then put these polymerase-loaded liposomes into a beaker of water in which two other molecules were floating. One was the nucleotide-- the watermelon. The other was protease, an even larger enzyme that acts like a molecular razor blade, cutting any other enzyme it meets into bits and pieces. (For anyone who works with enzymes, protease is a dirty word, says Chakrabarti.) They let the liposomes sit for three days, then added a dye that could seep through them and bind to RNA. In theory, if the nucleotide could slip through the pores of the membrane, it would be assembled into RNA by the polymerase. The dye would attach to the RNA and signal the researchers that the procedure had worked. The protease was too big to get inside the liposomes, but it would prevent any RNA from forming outside their protective walls.  We didn’t know if we’d see anything at all, says Chakrabarti. But we saw all these vesicles glowing red with RNA. I hadn’t expected it to be so dramatic. The liposomes had indeed allowed nucleotides to enter through their pores, and the polymerase had assembled them into RNA. The researchers thus showed that primordial liposomes forming in tide pools could have performed some essential cellular tricks.  As an analogy to early life, their quasi cell has obvious limits, Deamer and Chakrabarti know. It builds simplified RNA, using only one nucleotide rather than the full complement of four, and once the RNA is produced, it can’t do anything--it simply fills up the liposome. Joyce and Breaker, however, have the expertise necessary to take the quasi cell another step toward life. Over the past five years, they have perfected a method for making RNA evolve. Simply stated, they put loose RNA strands in a beaker and give them a job to do, such as cutting DNA; the ones that do the best are rewarded with offspring. The researchers place the selected RNA in a bath of loose nucleotides and enzymes and allow it to produce millions of copies of itself. They use this process to evolve the RNA by making the copying process slightly imperfect. Some variants do the designated task better than their ancestors, and they in turn are rewarded with progeny.  Deamer and his colleagues now want to put this whole loop of reactions inside their liposomes. There’s no guarantee it will work. They’ll have to sandwich the RNA, two enzymes, and two necessary primers in the lipids and hope that all five molecules get trapped in some of the liposomes. Then they’ll need to supply all four nucleotides in the water surrounding the liposomes and hope that the molecules can get into the liposomes fast enough to let the RNA direct its own reproduction.  Even if they succeed, many questions will remain before anyone will be able to build a functioning cell. How does it manage growth and division--a process that demands mind-boggling choreography even in a microbe? How exactly is this dance powered with energy? Yet there are far fewer questions to answer now than anyone expected. These things we’re now doing would have been unthinkable a few years ago, says Chakrabarti. It would be great one day to walk into the lab and say, ‘I think I’ll start up a cell today.’  I’m pretty sure in the next five or ten years we or somebody else will put together a system of molecules that can take a source of energy and make more of itself in an encapsulated environment, says Deamer. It’ll be technically alive, but if we put it out to compete in any natural environment, something will eat it long before it has a chance to make its way up the evolutionary ladder. It’s going to be a big deal when somebody gets to it, but this mystery of how genetic information came into the biosphere--that’s going to be unknown for years to come.  Deamer isn’t going to lose his patience, though. As we work our way toward the first living state, we find things all along the way. You pick a direction and start walking. That’s really what I’m doing. You may never know exactly how life began, but you’re going to learn a lot along the way.