CHAPTER 3: ROW, ROW, ROW YOUR BOAT

PROTEINS

As strange as it may seem, modern biochemistry has shown that the cell is operated by machines -- literally, molecular machines. Like their man-made counterparts (such as mousetraps, bicycles, and space shuttles), molecular machines range from the simple to the enormously complex: mechanical, force-generating machines, like those in muscles; electronic machines, like those in nerves; and solar-powered machines, like those of photosynthesis. Of course, molecular machines are made primarily of proteins, not metal and plastic. In this chapter I will discuss molecular machines that allow cells to swim, and you will see what is required for them to do so.

But first, some necessary details. In order to understand the molecular basis of life one has to have an idea of how proteins work. Those who want to know all the details -- how proteins are made, how their structures allow them to work so effectively, and so on -- are encouraged to borrow an introductory biochemistry textbook from the library. For those who want to know a few details -- such as what amino acids look like, and what are the levels of protein structure -- I have included an Appendix that discusses proteins and nucleic acids. For present purposes, however, an overview of these remarkable biochemicals will suffice.

Most people think of proteins as something you eat. In the body of a living animal or plant, however, they play very active roles. Proteins are the machines within living tissue that build the structures and carry out the chemical reactions necessary for life. For example, the first step in capturing the energy in sugar and changing it into a form the body can use is carried out by a catalyzing protein (also known as an enzyme) called hexokinase; skin is made up mostly of a protein called collagen; and when light strikes your retina, the protein called rhodopsin initiates vision. You can see even by this limited number of examples that proteins are amazingly versatile. Nonetheless, a given protein has only one or a few uses: rhodopsin cannot form skin, and collagen cannot interact usefully with light. Therefore a typical cell contains thousands and thousands of different kinds of proteins to perform the many tasks of life.

Proteins are made by chemically hooking together amino acids into a chain. A protein chain typically has anywhere from about fifty to about one thousand amino acid links. Each position in the chain is occupied by one of twenty different amino acids. In this they are like words, which can come in various lengths but are made up from a set of just 26 letters. As a matter of fact, biochemists often refer to each amino acid by a single-letter abbreviation -- G for glycine, S for serine, H for histidine, and so forth. Each different kind of amino acid has a different shape and different chemical properties. For example, W is large but A is small, R carries a positive charge but E carries a negative charge, S prefers to be dissolved in water but I prefers oil, and so on.

When you think of a chain, you probably think of something that is very flexible, without much overall shape. But chains of amino acids -- in other words, proteins -- aren't like that. Proteins that work in a cell fold up into very precise structures, and the structure can be quite different for different types of proteins. The folding is done automatically when, say, a positively charged amino acid attracts a negatively charged one, oil-preferring amino acids huddle together to exclude water, large amino acids are pushed out of small spaces, and so on. Two different amino acid sequences (that is two different proteins) can fold into structures as specific and different from each other as an adjustable wrench and a jigsaw.

It is the shape of a folded protein and the precise positioning of the different kinds of amino acid groups that allow a protein to work (Figure 3-1). For example, if it is the job of one protein to bind specifically to a second protein, then their two shapes must fit each other like a hand in a glove. If there is a positively charged amino acid on the first protein, then the second protein better have a negatively charged amino acid; otherwise, the two will not stick together. If it is the job of a protein to catalyze a chemical reaction, then the shape of the enzyme generally matches the shape of the chemical that is its target. When it binds, the enzyme has amino acids precisely positioned to cause a chemical reaction. If the shape of a wrench or a jigsaw is significantly warped, then the tool doesn't work. Likewise, if the shape of a protein is warped then it fails to do its job.

Modern biochemistry was launched forty years ago when science began to learn what proteins look like. Since then, great strides have been made in understanding exactly how particular proteins carry out particular tasks. In general, the cell's work requires teams of proteins; each member of the team carries out just one part of a larger task. To keep things as simple as possible, in this book I will concentrate on protein teams. Now, let's go swimming.

SWIMMING

Suppose, on a summer day, you find yourself taking a trip to the neighborhood pool for a bit of exercise. After slathering on the sunblock, you lie on a towel reading the latest issue ofNucleic Acids Researchand wait for the adult swim period to begin. When at long last the whistle blows and the overly energetic younger crowd clears the water, you gingerly dip your toes in. Slowly, painfully, you lower the rest of your body into the surprisingly cold water. Because it would not be dignified, you will not do any cannonballs or fancy dives from the diving board, nor play water volleyball with the younger adults. Rather, you will swim laps.

Pushing off from the side, you bring your right arm up over your head and plunge it into the water, completing one stroke. During the stroke, nerve impulses travel from your brain to your arm muscles, stimulating them to contract in a specific order. The contracting muscles tug against your bones, causing the humerus to rise and rotate. At the same time other muscles squeeze the bones of your fingers together, so that your hand forms a closed cup. Successive nerve impulses provoke other muscles to relax and contract, pulling in various ways on the radius and ulna, and directing the hand downward into the water. The force of the arm and hand on the water propel you forward. After completion of about half of the actions listed above a similar cycle begins, this time with the bones and muscles of the left arm. Simultaneously, nerve impulses travel to the muscles of your legs, causing them to contract and relax rhythmically, pulling the leg bones up and down. Slicing through the water at a stunning two miles per hour, though, you notice that it's getting hard to think; there's a burning sensation in your lungs; and, even though your eyes are open, things start to go black. Ah, yes -- you forgot to breathe. It was said of President Ford that he couldn't walk and chew gum at the same time; you find it difficult to coordinate the turning of your head to the water's surface and back again with the other motions required for swimming. Without oxygen to metabolize fuel your brain starts to shut down, preventing conscious nerve impulses from traveling to the distant regions of your body.

Before you pass out and suffer the humiliation of being rescued by a Generation X lifeguard you stop, stand up in the four feet of water, and notice that you're only about twenty feet from the side. To get around the breathing problem, you decide to do the backstroke. The backstroke involves most of the same muscles as freestyle swimming, and allows you to breathe without coordinating neck muscles with everything else. But now you can't see where you're going. Inevitably you drift off course, come too close to the volleyball game, and are smacked in the head by an errant overhand smash.

In order to get far away from the apologetic volleyballers, you decide simply to tread water in the deep end of the pool. Treading water uses your leg muscles, giving you the exercise you want. It also allows both easy breathing and clear vision. After a few minutes, however, your legs begin to cramp. Deep inside your flabby limbs, unknown to you, your seldom-used muscles keep on hand enough fuel for only short bursts of activity, followed by long periods of rest. During the unusually prolonged exercise they quickly run out of sustenance and cease to function effectively. Nerve impulses frantically try to provoke the motions necessary for swimming, but with the muscles malfunctioning, your legs are as useless as a mousetrap with a broken spring.

You relax and remain still. Fortunately, the large region of your body around the waist has a density less than that of water, and so it keeps you afloat. After a minute or two of bobbing in the water, your cramped muscles relax. You spend the rest of the adult swim period floating serenely around the deep end. This doesn't provide much exercise, but at least it is enjoyable -- until the whistle blows again, and you are pummeled by the cannonballs of undignified kids.

WHAT IT TAKES

The neighborhood pool scenario illustrates the requirements for swimming. it also shows that efficiency can be improved by adding auxiliary systems to the basic swimming equipment. To take the last scene first, floating requires only that an object be less dense than water; it does not require activity. The ability to float -- to be able to keep a portion of the body out of the water with no active effort -- can certainly be useful. Yet because the floater simply drifts along with the current, the ability to float is not the same thing as the ability to swim.

A direction-finding system (such as eyesight) is also useful for swimming; however, it is not the same thing as the ability to swim. In the story you could do the backstroke for a while and still advance through the water. Eventually, an inability to sense the surroundings can lead to accidents. Nonetheless, one can swim sighted or one can swim blind.

Swimming clearly requires energy; cramped, useless muscles immediately cause the system to fail. But you traveled twenty feet before running out of oxygen, and then treaded water for a short while before cramping set in. Although they certainly affect the distance a swimmer can go, the size and efficiency of the fuel reserve system thus are not parts of the swimming system itself.

Now let's consider the mechanical requirements of swimming. You used your hands and feet to contact the water and push it, thus moving your body in the opposite direction. Without the limbs, or some substitute, active swimming would be quite impossible. So we can conclude that one requirement for swimming is a paddle. Another requirement is a motor or power source that has at least enough fuel to last several cycles. At the organ level in humans, the motor is the leg or arm muscle that alternately contracts and relaxes. If the muscle is paralyzed; there is no effective motor, and swimming is impossible. The final requirement is for a connection between the motor and the paddling surface: in humans, these are the areas of bones to which the muscles adhere. If a muscle is detached from a bone it can still contract; because it does not move the bone, however, swimming does not take place.

Mechanical examples of swimming systems are easy to find. My youngest daughter has a toy wind-up fish that wiggles its tail, propelling itself somewhat awkwardly through the bathtub. The tail of the toy fish is the paddle surface, the wound spring is the energy source, and a connecting rod transmits the energy. If one of the components -- the paddle, motor, or connector -- is missing, then the fish goes nowhere. Like a mousetrap without a spring, a swimming system without a paddle, motor, or connector is fatally incomplete. Because the swimming systems need several parts to work, they are irreducibly complex.

Keep in mind that we are discussing only the parts common to all swimming systems -- even the most primitive. Additional complexity is frequently seen. For example, my daughter's toy fish has, besides its tail, spring, and connecting rod, several gears that transmit force from the rod to the tail. A propeller-driven ship has all manner of gears and rods redirecting the energy of the motor until it is finally transmitted to the propeller. Unlike the eye of a swimmer, which is separate from the swimming system itself, such extra gears are indeed part of the system -- removing them causes the whole setup to grind to a halt. When a real-life system has more than the theoretically minimum number of parts, then you have to check each of the other parts to see if they're required for the system to work.

WHAT ELSE IT TAKES

A simple list of pieces shows the very minimum of requirements. In the last chapter I discussed how a mousetrap that had all the necessary pieces -- a hammer, base, spring, catch, and holding bar -- still might not work. If the holding bar were too short or the spring too lightweight, for example, the trap would be a failure. Similarly, the pieces of a swimming system must be matched to each other to have at least minimal function. The paddle is necessary, but if its surface is too small a boat might not make enough progress in a required amount of time. Conversely, if the paddle surface is too large, the connector or motor might strain and break when moving. The motor must be strong enough to move the paddle. It must also be regulated to go at an appropriate speed: too slow, and the swimmer does not make physically necessary progress; too fast, and the connector or paddle may break.

But even if we have the right parts of a swimming system, and even if the parts are the right size and strength and are matched to each other, more is needed. The additional requirement -- the need to control the timing and direction of the paddle strokes -- is easier to see in the example of a human swimmer than in the case of a paddleboat. When a nonswimmer falls into the water he helplessly flails his arms and legs, making no more progress than if he simply floated. Even a beginning swimmer like my oldest daughter, who is just learning the strokes, quickly sinks unless Dad supports her. Her individual strokes are adequate, but their timing is not coordinated, she doesn't hold herself parallel to the water's surface, and she keeps her head out of the water.

Mechanical systems seem not to have those problems. A ship doesn't flail its propeller, and the timing and direction of a paddleboat's strokes are smooth and regular from the beginning. But the argument is deceptive. The apparently effortless abilities are actually built into the shape and connectivity of the paddlewheel, rotor, and motor of the boat. imagine a steamboat in which the paddle boards were not arranged nicely around a circular frame. Suppose the boards went off at various angles and the rotor turned first forward, then backward, then side to side. Instead of taking a scenic tour of the Mississippi the boat would drift helplessly, spastically floating with the current toward the Gulf of Mexico. A propeller with blades set at haphazard angles would churn water, but it wouldn't move a boat in any particular direction. The apparent ease with which a mechanical system paddles -- compared to the difficulties of a human non-swimmer -- is an illusion. The engineer who designed the system "trained" it to swim, pushing the water in the correct direction with the correct timing.

In the unforgiving world of nature, an organism spending energy to flail helplessly in the water would have no advantage over the organism floating serenely beside it. Do any cells swim? If so, what swimming systems do they use? Are they, like a Mississippi steamboat, irreducibly complex? Could they have evolved gradually?

THE CILIUM

Some cells swim using a cilium. A cilium is a structure that, crudely put, looks like a hair and beats like a whip. if a cell with a cilium is free to move about in a liquid, the cilium moves the cell much as an oar moves a boat. If the cell is stuck in the middle of a sheet of other cells, the beating cilium moves liquid over the surface of the stationary cell. Nature uses cilia for both jobs. For example, sperm use cilia to swim. in contrast, the stationary cells that line the respiratory tract each have several hundred cilia. The large number of cilia beat in synchrony, much like the oars handled by slaves on a Roman galley ship, to push mucus up to the throat for expulsion. The action removes small foreign particles -- like soot -- that are accidentally inhaled and stick in the mucus.

Light microscopes showed thin hairs on some cells, but discovery of the Lilliputian details of cilia had to wait for the invention of the electron microscope, which revealed that the cilium is quite a complicated structure. I will be discussing the structure of the cilium for the next few pages. Most readers will probably find the discussion easier to follow by referring frequently to Figure 3-2.

The cilium consists of a membrane-coated bundle of fibers. The ciliary membrane (think of it as a sort of plastic cover) is an outgrowth of the cell membrane, so the interior of the cilium is connected to the interior of the cell. When a cilium is sliced crossways and the cut end is examined by electron microscopy, you see nine rod-like structures around the periphery. The rods are called microtubules. When high-quality photographs are closely inspected, each of the nine microtubules is seen to actually consist of two fused rings. Further examination shows that one of the rings is made from thirteen individual strands. The other ring, joined to the first, is made from ten strands. Summarizing briefly, each of the nine outer microtubules of a cilium is made of a ring of ten strands fused to a ring of thirteen strands.

Biochemical experiments show that microtubules are made from a protein called tubulin. In a cell, tubulin molecules come together like bricks that form a cylindrical smokestack. Each of the nine outer rods is a microtubule that resembles a fused, double-smokestack with bricks of tubulin. Pictures produced by electron microscopy also show two rods in the middle of the cilium. They, too, are microtubules. Instead of being double smokestacks, however, they are individual smokestacks, each made of thirteen strands of tubulin.

When conditions are right within the cell (for example, when the temperature is within certain limits and when the concentration of calcium is just right), tubulin -- the "brick" that makes up the smokestacks -- automatically comes together to form microtubules. The forces that bring tubulins together are much like those that fold an individual protein into a compact shape: positive charges attract negative charges, oily amino acids squeeze together to exclude water, and so forth. One end of a tubulin molecule has a surface that is complementary to the opposite end of a second tubulin molecule, so the two stick together. A third tubulin can then stick onto the end of the second molecule, a fourth onto the end of the third, and so on. As an analogy, think of the stacking of tuna cans. In the grocery store where my family shops the tuna cans, because the bottom is beveled and is the same diameter as the straight-edged top, stack snugly one on top of the other. If the stack is gently bumped, the cans remain in place.

If two tuna cans are stacked top-to-top instead of top-to-bottom, though, they do not stack securely and can be moved by a casual bump. Furthermore, if Brand X tuna does not have a beveled bottom, it does not stack securely on itself because its cans do not have complementary surfaces. The association of tubulin molecules is much more specific than the stacking of tuna cans, After all, in the cell there are thousands of different proteins, and tubulin has to be sure to associate only with other tubulins -- not with just any protein that comes along. Perhaps, then, we should think of tubulin as a tuna can with ten short needle-like projections distributed over the top surface, and ten indentations in the bottom that exactly match the positions of the projections on the top. Now no tuna can will accidentally stack with any other type of can.

Extending our tuna analogy, suppose we also had several projections sticking out one side of the can that were complementary to indentations located almost, but not quite, on the exact opposite side. Then we could stick the cans together side by side and, because the holes were not quite opposite the projections, when we put more cans together they would eventually circle around and form a closed loop. Stacking loops upon loops we eventually (after thoroughly mixing our metaphors) make a structure like a smokestack from our tuna cans.

Although tubulin has the power to self-associate into microtubules, microtubules do not aggregate with one another without help from other proteins. There is a good reason for this: microtubules have a number of jobs to do in the cell. For most jobs, single, unassociated microtubules are needed. For other jobs (including ciliary motion), however, bundles of microtubules are needed. So microtubules lie around individually, like the rods from the game of pick-up sticks, unless purposely bundled together for a particular job.

In photographs of cilia taken by an electron microscope, several different types of connectors can be seen tying together the individual microtubules (see Figure 3-2). There is a protein that bridges the two central single microtubules in the middle of the cilium. Also, from each of the double microtubules, a radial spoke projects toward the center of the cilium. The structure ends in a knobby mass called the spoke head. Finally, a protein called nexin connects each outer, double microtubule to the one beside it.

Two other projections adorn each peripheral microtubule; they are called the outer arm and the inner arm. Biochemical analysis has shown that these projections contain a protein called dynein. Dynein is a member of a class of proteins called motor proteins, which function as tiny motors in the cell, powering mechanical motion.

HOW A CILIUM WORKS

Knowing the structure of a complex machine and knowing how it works are two different matters. One could open the hood of a car and take pictures of the motor until the cows come home, but the snapshots by themselves would not give a clear idea of how the different parts produced the function. Ultimately, in order to find out how a thing works, you have to take it apart and reassemble it, stopping at many points to see if function has yet been restored. Even this may not yield a clear idea of how the machine operates, but it does give a working knowledge of which components are critical. The basic strategy of biochemistry in this century has been to take apart molecular systems and try to put them back together. The strategy has yielded enormous insights into the operations of the cell.

Experiments of this sort have given biochemists clues to how the cilium works. The first clue comes from isolated cilia. Nature has kindly arranged it so that cilia can be separated from cells by vigorous shaking. The shaking breaks off the projections cleanly and, by spinning the solution at high speed (which causes big, heavy particles to sediment more quickly than small, light particles), one can obtain a solution of pure cilia in a test tube. If the cilia are stripped of their membrane and then supplied with a chemical form of energy called ATP, they will beat in characteristic whip-like fashion. This result shows that the motor to power ciliary motion resides in the cilium itself -- not in the interior of the now-missing cell. The next clue is that if (through biochemical tricks) the dynein arms are removed but the rest of the cilium is left intact, then the cilium is paralyzed, as if in rigor mortis. Adding back fresh dynein to the stiffened cilia allows motion to resume. So it appears that the motor of the cilium is contained in the dynein arms.

Further experiments gave more clues. There are enzymes (called proteases) that have the ability to chew up other proteins, decomposing them into amino acids. When a small amount of a protease is added for a short time to a solution containing cilia, the protease quickly slices up the nexin linkers at the edge of the structure. The rest of the cilium remains intact. The reason that the protease rapidly attacks the linkers is that, unlike the other proteins of the cilium, the nexin linkers are not folded up tightly; instead, they are loose, flexible chains. Because they are loose, the protease can cut them as rapidly as a pair of scissors can cut a paper ribbon. (The protease cuts tightly folded proteins as rapidly as scissors cut a closed paperback book.)

Proteases allowed biochemists to see how a cilium would work without nexin linkers. What would removal of the linkers do? Perhaps the cilium would work just fine without them, or perhaps it would go into rigor mortis as it did when the dynein arms were removed. In fact, neither of these possibilities occurred. Instead, the linkerless cilium did something quite unexpected. When biochemical energy was supplied to the cilium, instead of bending, it rapidly unraveled. The individual microtubules began to slide past one another like the segments of a radio antenna slide past one another when it is opened. They continued to slide until the length of the cilium had increased by almost tenfold. From this result biochemists concluded that the motor was working, since something had to move the individual microtubules. They also concluded that the nexin linkers are needed to keep the cilium together when it is trying to bend.

These clues have led to a model for how the cilium works (see Figure 3-2). Imagine several smokestacks made of tuna cans that are tightly held together. The tuna can smokestacks are connected by slack wires. Attached to one smokestack is a little motor with an arm that reaches out and holds on to a tuna can in a neighboring smokestack. The motor arm pushes the second smokestack down, sliding it past the first one. As the smokestacks slide past each other, the slack wires begin to stretch and become taut. As the motor arm pushes more, the strain from the wire makes the smokestacks bend. Thus the sliding motion has been converted into a bending motion. Now, let's translate the analogy into biochemical terms. The dynein arms on one microtubule attach to a second, neighboring microtubule, and the dynein uses the biological energy of ATP to "walk up" its neighbor. When this happens the two microtubules begin to slide past each other. In the absence of nexin, they would continue to slide until they separated; however, the protein cross-links prevent neighboring microtubules from sliding by more than a short distance. When the flexible nexin linkers have been elongated to their limit, further walking by dynein makes the nexin linkers tug on the microtubules. As dynein continues its walk, strain increases. Fortunately the microtubules are somewhat flexible, so the dynein-induced sliding motion is converted to a bending motion.

Now, let us sit back, review the workings of the cilium, and consider what they imply. What components are needed for a cilium to work? Ciliary motion certainly requires microtubules; otherwise, there would be no strands to slide. Additionally it requires a motor, or else the microtubules of the cilium would lie stiff and motionless. Furthermore, it requires linkers to tug on neighboring strands, converting the sliding motion into a bending motion, and preventing the structure from falling apart. All of these parts are required to perform one function: ciliary motion. Just as a mousetrap does not work unless all of its constituent parts are present, ciliary motion simply does not exist in the absence of microtubules, connectors, and motors. Therefore we can conclude that the cilium is irreducibly complex -- an enormous monkey wrench thrown into its presumed gradual, Darwinian evolution.

The fact that the cilium is irreducibly complex should surprise no one. Earlier in this chapter we saw that a swimming system requires a paddle to contact the water, a motor or source of energy, and a connector to link the two. All systems that move by paddling -- ranging from my daughter's toy fish to the propeller of a ship -- fail if any one of the components is absent. The cilium is a member of this class of swimming systems. The microtubules are the paddles, whose surface contacts the water and pushes against it. The dynein arms are the motors, supplying the force to move the system. The nexin arms are the connectors, transmitting the force of the motor from one microtubule to its neighbor.

The complexity of the cilium and other swimming systems isinherent in the task itself.it does not depend on how large or small the system is, whether it has to move a cell or move a ship: in order to paddle, several components are required. The question is, how did the cilium arise?

AN INDIRECT ROUTE

Some evolutionary biologists -- like Richard Dawkins -- have fertile imaginations. Given a starting point, they almost always can spin a story to get to any biological structure you wish. The talent can be valuable, but it is a two-edged sword. Although they might think of possible evolutionary routes other people overlook, they also tend to ignore details and roadblocks that would trip up their scenarios. Science, however, cannot ultimately ignore relevant details, and at the molecular level all the "details" become critical. If a molecular nut or bolt is missing, then the whole system can crash. Because the cilium is irreducibly complex, no direct, gradual route leads to its production. So an evolutionary story for the cilium must envision a circuitous route, perhaps adapting parts that were originally used for other purposes. Let's try, then, to imagine a plausible indirect route to a cilium using pre-existing parts of the cell.

To begin, microtubules occur in many cells and are usually used as mere structural supports, like girders, to prop up cell shape. Furthermore, motor proteins also are involved in other cell functions, such as transporting cargo from one end of the cell to another. The motor proteins are known to travel along microtubules, using them as little highways to get from one point to another. An indirect evolutionary argument might suggest that at some point several microtubules stuck together, maybe to reinforce some particular cell shape. After that, a motor protein that normally traveled on microtubules might have accidentally acquired the ability to push two neighboring microtubules, causing a slight bending motion that somehow helped the organism survive. Further small improvements gradually produced the cilium we find in modern cells.

Intriguing as this scenario may sound, though, critical details are overlooked. The question we must ask of this indirect scenario is one for which many evolutionary biologists have little patience: but howexactly?

For example, suppose you wanted to make a mousetrap. In your garage you might have a piece of wood from an old Popsicle stick (for the platform), a spring from an old wind-up clock, a piece of metal (for the hammer) in the form of a crowbar, a darning needle for the holding bar, and a bottle cap that you fancy to use as a catch. But these pieces couldn't form a functioning mousetrap without extensive modification, and while the modification was going on, they would be unable to work as a mousetrap. Their previous functions make them ill-suited for virtually any new role as part of a complex system.

In the case of the cilium, there are analogous problems. The mutated protein that accidentally stuck to microtubules would block their function as "highways" for transport. A protein that indiscriminately bound microtubules together would disrupt the cell's shape -- just as a building's shape would be disrupted by an erroneously placed cable that accidentally pulled together girders supporting the building. A linker that strengthened microtubule bundles for structural supports would tend to make them inflexible, unlike the flexible linker nexin. An unregulated motor protein, freshly binding to microtubules, would push apart microtubules that should be close together. The incipient cilium would not be at the cell surface. If it were not at the cell surface, then internal beating could disrupt the cell; but even if it were at the cell surface, the number of motor proteins would probably not be enough to move the cilium. And even if the cilium moved, an awkward stroke would not necessarily move the cell. And if the cell did move, it would be an unregulated motion using energy and not corresponding to any need of the cell. A hundred other difficulties would have to be overcome before an incipient cilium would be an improvement for the cell.

SOMEBODY MUST KNOW

The cilium is a fascinating structure that has intrigued scientists from many disciplines. The regulation of its size and structure interests biochemists; the dynamics of its power stroke fascinate biophysicists; the expression of the many separate genes coding for its components engrosses the minds of molecular biologists. Even physicians study them, because cilia are medically important: they occur in some infectious microorganisms, and cilia in the lungs get clogged in the genetic disease cystic fibrosis. A quick electronic search of the professional literature shows more than a thousand papers in the past several years that haveciliaor a similar word in the title. Papers have appeared on related topics in almost all the major biochemistry journals, includingScience, Nature, Proceedings of the National Academy of Sciences, Biochemistry, Journal of Biological Chemistry, Journal of Molecular Biology, Cell,and numerous others. in the past several decades, probably ten thousand papers have been published concerning cilia.

Since there is such a large literature on the cilium, since it is of interest to such diverse fields, and since it is widely stated that the theory of evolution is the basis of all modern biology, then one would expect that the evolution of the cilium would be the subject of a significant number of papers in the professional literature. One might also expect that, although perhaps some details would be harder to explain than others, on the whole science should have a good grasp of how the cilium evolved. The intermediate stages it probably went through, the problems that it would encounter at early stages, the possible routes around such problems, the efficiency of a putative incipient cilium as a swimming system -- all of these would certainly have been thoroughly worked over. In the past two decades, however, only two articles even attempted to suggest a model for the evolution of the cilium that takes into account real mechanical considerations. Worse, the two papers disagree with each other even about the general route such an evolution might take. Neither paper discusses crucial quantitative details, or possible problems that would quickly cause a mechanical device such as a cilium or a mousetrap to be useless.

The first paper, authored by T. Cavalier-Smith, appeared in 1978 in a journal calledBioSystems.The paper does not try to present a realistic quantitative model for even one step in the development of a cilium in a cell line originally lacking that structure. Instead it paints a picture of what the author imagines must have been significant events along the way to a cilium. These imaginary steps are described in phrases such as "flagella [long cilia are frequently called "flagella"] are so complex that their evolution must have involved many stages"; "I suggest that flagella initially need not have been motile, but were slender cell extensions"; "organisms would evolve with a great variety of axonernal structures"; and "it is likely that mechanisms of phototaxis [motion toward light] evolved simultaneously with flagella."

The quotations give the flavor of the fuzzy word-pictures typical of evolutionary biology. The lack of quantitative details -- a calculation or informed estimation based on a proposed intermediate structure of how much any particular change would have improved the active swimming ability of the organism -- makes such a story utterly useless for understanding how a cilium truly might have evolved.

Let me hasten to add that the author (a well-known scientist who has made a number of important contributions to cell biology) didn't intend that the paper should be taken as presenting a realistic model; he was just trying to be provocative. He was hoping to entice other workers with the promise of his model, however vaguely constructed -- to goad them into doing some work to flesh out the emaciated skeleton. Such provocation can be an important service in science. Unfortunately, in the intervening years no one has built upon the model.

The second paper, authored nine years later by a Hungarian scientist named Eörs Szathmary and also appearing inBioSystems,is similar in many ways to the first paper. Szathmary is an advocate of the idea, championed by Lynn Margulis, that cilia resulted when a type of swimming bacterium called a "spirochete" accidentally attached itself to a eukaryotic cell. The idea faces the considerable difficulty that spirochetes move by a mechanism (described later) that is totally different from that for cilia. The proposal that one evolved into the other is like a proposal that my daughter's toy fish could be changed, step by Darwinian step, into a Mississippi steamboat. Margulis herself is not concerned with mechanical details; she is content to look for general similarities in some components of cilia and bacterial swimming systems. Szathmary attempted to go a little further and actually discuss mechanical difficulties that would have to be overcome in such a scenario. Inevitably, however, his paper (like Cavalier-Smith's) is a simple word-picture that presents an underdeveloped model to the scientific community for further work. It also has failed at provoking such experimental or theoretical work, either by the author or by others.

Margulis and Cavalier-Smith have clashed in print in recent years. Each points out the enormous problems with the other's model, and each is correct. What is fatal, however, is that neither side has filled in any mechanistic details for its model. Without details, discussion is doomed to be unscientific and fruitless. The scientific community at large has ignored both contributions; neither paper has been cited by other scientists more than a handful of times in the years since publication.

The amount of scientific research that has been and is being done on the cilium -- and the great increase over the past few decades in our understanding of how the cilium works -- lead many people to assume that even if they themselves don't know how the cilium evolved, somebody must know. But a search of the professional literature proves them wrong. Nobody knows.

THE BACTERIAL FLAGELLUM

We humans tend to have a rather exalted opinion of ourselves, and that attitude can color our perception of the biological world. In particular, our attitude about what is higher and lower in biology, what is an advanced organism and what is a primitive organism, naturally starts with the presumption that the pinnacle of nature is ourselves. The presumption can be defended by citing human dominance, and also with philosophical arguments. Nonetheless, other organisms, if they could talk, could argue strongly for their own superiority. This includes bacteria, which we often think of as the rudest forms of life.

Some bacteria boast a marvelous swimming device, the flagellum, which has no counterpart in more complex cells. In 1973 it was discovered that some bacteria swim by rotating their flagella. So the bacterial flagellum acts as a rotary propeller -- in contrast to the cilium, which acts more like an oar.

The structure of a flagellum (Figure 3-3) is quite different from that of a cilium. The flagellum is a long, hairlike filament embedded in the cell membrane. The external filament consists of a single type of protein, called "flagellin." The flagellin filament is the paddle surface that contacts the liquid during swimming. At the end of the flagellin filament near the surface of the cell, there is a bulge in the thickness of the flagellum. It is here that the filament attaches to the rotor drive. The attachment material is comprised of something called "hook protein." The filament of a bacterial flagellum, unlike a cilium, contains no motor protein; if it is broken off, the filament just floats stiffly in the water. Therefore the motor that rotates the filament-propeller must be located somewhere else. Experiments have demonstrated that it is located at the base of the flagellum, where electron microscopy shows several ring structures occur. The rotary nature of the flagellum has clear, unavoidable consequences, as noted in a popular biochemistry textbook:

[The bacterial rotary motor] must have the same mechanical elements as other rotary devices: a rotor (the rotating element) and a stator (the stationary element.)

The rotor has been identified as the M ring in Figure 3-3, and the stator as the S ring.

The rotary nature of the bacterial flagellar motor was a startling, unexpected discovery. Unlike other systems that generate mechanical motion (muscles, for example) the bacterial motor does not directly use energy that is stored in a "carrier" molecule such as ATP. Rather, to move the flagellum it uses the energy generated by a flow of acid through the bacterial membrane. The requirements for a motor based on such a principle are quite complex and are the focus of active research. A number of models for the motor have been suggested; none of them are simple. (One such model is shown in Figure 3-3 just to give the reader a taste of the motor's expected complexity.)

The bacterial flagellum uses a paddling mechanism. Therefore it must meet the same requirements as other such swimming systems. Because the bacterial flagellum is necessarily composed of at least three parts -- a paddle, a rotor, and a motor -- it is irreducibly complex. Gradual evolution of the flagellum, like the cilium, therefore faces mammoth hurdles.

The general professional literature on the bacterial flagellum is about as rich as the literature on the cilium, with thousands of papers published on the subject over the years. That isn't surprising; the flagellum is a fascinating biophysical system, and flagellated bacteria are medically important. Yet here again, the evolutionary literature is totally missing. Even though we are told that all biology must be seen through the lens of evolution, no scientist has ever published a model to account for the gradual evolution of this extraordinary molecular machine.

IT ONLY GETS WORSE

Above I noted that the cilium contains tubulin, dynein, nexin, and several other connector proteins. If you take these and inject them into a cell that lacks a cilium, however, they do not assemble to give a functioning cilium. Much more is required to obtain a cilium in a cell. A thorough biochemical analysis shows that a cilium contains overtwo hundred different kinds of proteins;the actual complexity of the cilium is enormously greater than what we have considered. All of the reasons for such complexity are not yet clear and await further experimental investigation. Other tasks for which the proteins might be required, however, include attachment of the cilium to a base structure inside the cell; modification of the elasticity of the cilium; control of the timing of the beating; and strengthening of the ciliary membrane.

The bacterial flagellum, in addition to the proteins already discussed, requires about forty other proteins for function. Again, the exact roles of most of the proteins are not known, but they include signals to turn the motor on and off; "bushing" proteins to allow the flagellum to penetrate through the cell membrane and cell wall; proteins to assist in the assembly of the structure; and proteins to regulate the production of the proteins that make up the flagellum.

In summary, as biochemists have begun to examine apparently simple structures like cilia and flagella, they have discovered staggering complexity, with dozens or even hundreds of precisely tailored parts. It is very likely that many of the parts we have not considered here are required for any cilium to function in a cell. As the number of required parts increases, the difficulty of gradually putting the system together skyrockets, and the likelihood of indirect scenarios plummets. Darwin looks more and more forlorn. New research on the roles of the auxiliary proteins cannot simplify the irreducibly complex system. The intransigence of the problem cannot be alleviated; it will only get worse. Darwinian theory has given no explanation for the cilium or flagellum. The overwhelming complexity of the swimming systems push us to think it may never give an explanation.

As the number of systems that are resistant to gradualist explanation mounts, the need for a new kind of explanation grows more apparent. Cilia and flagella are far from the only problems for Darwinism. in the next chapter I will look at the biochemical complexity underlying the apparent simplicity of blood clotting.

Excerpted from Darwin's Black Box: The Biochemical Challenge to Evolution by Michael J. Behe
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