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.
. The cilium consists of a membrane-coated bundle of fibers.1 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
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.
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-in-duced 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.2
The complexity of the cilium and other swimming systems is inherent
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. Further-
more, 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 how exactly?
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 have
cilia or a similar word in the title. Papers have appeared on related topics
in almost all the major biochemistry journals, including Science, 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 called BioSystems.3 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»; «l suggest that flagella
initially need not have been motile, but were slender cell extensions»;
«organisms would evolve with a great variety of axonemal 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 in BioSystems, is
similar in many ways to the first paper.4 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.5 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.6
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.7
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.
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.8 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
. The structure of a flagellum 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
[The bacterial rotary motor] must have the same mechanical elements
as other rotary devices: a rotor (the rotating element) and a stator (the
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 over two
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.
Read Darwin’s Black Box, Michael Behe’s book.
God bless you/Deus te abençoe