Eyelashes Up Close
Cilia are a must-have appendage, and they do much more than swat bodily currents—indeed, new research is suggesting they play an important role in development and disease.
When Antonie van Leeuwenhoek looked down at a prokaryote through his simple microscope made of a single mounted lens back in the 1660s, he discovered the first organelle. Captivated by the fluttering “legs” that would later be called the cell’s eyelashes, or cilia in Latin, he might have wondered about the origin of their movement.
That mystery remained unsolved even into the late 1950s when I started working as a graduate student in Keith Porter’s lab at the Rockefeller Institute for Medical Research (now Rockefeller University). Porter had made important discoveries on cilia. After writing my senior undergraduate thesis on the organelle, I was thrilled with the idea of working in his lab and tackling the question of motility for myself.
A few years before I joined the lab, Porter had pioneered methods that allowed us to use the electron microscope to study cellular structures. It revealed pictures of the internal organization of a cell organelle as small as a cilium at high resolution. The pictures revealed that the cilium, which is only about 250 nanometers in diameter (despite sometimes being many millimeters in length) contained filaments arranged in nine matching petals, each made of two fused filaments, around a central pair that isn’t fused, collectively called the axoneme.
It was such an intriguing and well-conserved pattern that I couldn’t help but think that something about it would reveal how the cilium moved. But after getting nowhere with the problem for several years, I took a break, moving to Copenhagen to work on unrelated research.
Researchers had known that properly moving cilia were not only important to protozoans swimming in their dish, but also for the proper function of the ciliated cells that comprised organs: from the trachea to the lining of the brain and the female reproductive tract, and there was more to come.1 We soon realized that the flashier variety of cilia—the kind that moved—was only a small part of the story. Cilia with no ability to move studded cells of sensory organs like the eye or the insect ear. When I returned from Copenhagen, researchers began to point out that many ordinary tissue cells also bore non-moving single cilia.
Only recently did we discover that those cilia were actually crucial as signaling appendices, acting as cells’ antennae. They have since opened up many more questions regarding the evolution and function of this intriguing organelle.
In the 1960s, evidence was beginning to accumulate that the axoneme, with its nine filaments plus the two in the center (9+2), powered by ATP, was responsible for the motility of the cilium, but it was unclear how the energy was harnessed and which molecules of the axoneme were involved in the production and control of motility.
After I returned from Copenhagen, I took up the question of motility again. The challenge was that the cilia beat as fast as 100 times per second and when stopped with a fixative, they all locked into the same straightened shape. The break came when I found that I could use osmium tetroxide to fix the cilia in the act of moving, as they swung from one side to another. (I didn’t realize that this technique was already in the literature and thought that I had invented it.)
Most researchers of the day assumed that ATP caused the filaments to contract on one side of the axoneme, making the entire structure bend in the direction of the contraction. Looking at some of the electron microscopy photos of the cross-sectioned tip of the cilia, I realized that if we captured the tip of the axoneme as it swayed in one direction, we should be able to see the filaments on the inner bend disappear as they contracted.
When I moved to the University of Chicago and became an assistant professor, I took multiple electron-microscopic photos of the cilium tip as it moved, creating a flip-book of the filaments to capture their position in motion. Surprisingly, rather than disappearing on the inner bend, the filaments on the opposite side disappeared.
The story was beginning to come together in my mind. In 1965, Ian Gibbons had described a new kind of protein attached to each of the ciliary filaments. It was a polymer that studded the length of each doublet. He named it dynein, for force (dyne) and protein (in), to explain his observation that these proteins were activated by ATP. With the results collected, I moved to the University of California, Berkeley in 1967 with a few more analyses to complete. Certain that the filaments, now called microtubules, couldn’t contract, and with a very plausible alternative explanation, I incorporated Gibbons’s finding and hypothesized the sliding microtubule model of ciliary motility.2 The model proposed that as the cilium moved, the microtubules did not change their lengths but slid past each other to accommodate the developing bend. This explained why we saw filaments disappear on the opposite side of the bend—the filaments slid in opposite directions, leaving one filament per pair at the tip, and curving the axoneme.
When I first showed my flip-book images to my colleagues, I saw their faces turn to expressions of both skepticism and surprise. It was somewhat heretical to propose a hypothesis that disputed the contractile theory in our own field. But the model of sliding filaments was a well-accepted mechanism in another area: muscle fiber contraction.
In the next few years, Gibbons’s lab provided new support for the model. They showed that when ATP was added to axonemes that had been cut from a cell, some of the microtubules slip out of the cilium. Using the electron microscope, my student Win Sale and I then demonstrated that to produce the movement, one of the microtubule doublets walks or slides toward the body of the cell on dynein feet, pushing its neighbor toward the tip of the cilium.
In the years that followed, it became clear that cilia were far more important for normal physiology than we had initially presumed. In the 1970s, Bjorn Afzelius from the University of Stockholm in Sweden showed that missing dynein arms led to immotility of human sperm, resulting in infertility. Similarly, mutations in various specific dyneins in respiratory cilia lead to a genetic respiratory disease, now called primary ciliary dyskinesia (PCD). Most surprisingly, Afzelius postulated that missing dyneins in cilia that must beat in the embryonic node during gastrulation were responsible for situs inversus, where the heart is on the right side of the body, rather than the left, and other organs are also reversed in left–right position. In the 1990s, Afzelius’s postulate was proven to be correct.
With the publication of studies linking cilia to human health and disease, I felt a renewed sense of interest in this organelle. Little did I know that we were only looking at the smaller part of the story.
In the late 1990s, researchers started to pay more attention to another family of cilia, the long-neglected solitary nonmotile cilia of ordinary cells, called primary cilia, that not only appeared on epithelial cells (for example, in the kidney tubule), but also smooth muscle cells, fibroblasts, neurons, and Schwann cells. The obvious difference between the motile and nonmotile cilia was that the central pair of microtubules was missing from the ciliary axoneme, making the axonemal pattern 9+0—like a flower without its pistil. Erroneously, we thought that the central pair of microtubules was critical for motility. (More recent research has revealed that the central pair of microtubules is necessary for the most efficient motion, rather than motion per se.) These cilia were considered a vestigial component of cells that no longer required movement. For about 20 years, primary cilia disappeared as an object of interest and were left out of major cell biology textbooks.
How wrong this was! More than any other aspect of our research, it is the primary cilium with its implications for development, physiology, and biomedicine that has revitalized the field. Joel Rosenbaum’s group at Yale University was studying how the cilium was built using Chlamydomonas, a single celled green alga with motile cilia (historically called flagella in this organism), as a model.3 Cilia growth was a complex process involving transport of materials from the cell body to the growing tip of the cilium. With Rosebaum’s group, George Witman and Greory Pazour at the University of Massachusetts Medical Center discovered that the same process that built the Chlamydomonas cilium built all cilia in the body, including the 9+0 primary cilia of kidney tubule cells, for example. In addition, mutations in a protein that stopped the growth of the Chlamydomonas cilium, affected a similar protein in the kidney, stunting the primary cilia, and giving rise to a form of polycystic kidney disease (PKD). With implications for a major human disease, primary cilia suddenly appeared on everyone’s radar screen.
The 9+0 axoneme and membrane are excellent substrates on which to build a cell that will respond to defined sensory stimuli: It extends into the extracellular matrix like an antenna in a privileged position to receive a stimulus, and internally localizes and concentrates growth factors or other proteins for signaling to the nucleus. My group continued to work on motile cilia, but we had started to look at how motile cilia responded to external stimuli through membrane receptors that generated messenger signals. What everyone learned from the analysis of kidney cilia and PKD was that signaling was a common feature of both motile and primary cilia.
When my postdoctoral fellow Søren Christensen returned to the University of Copenhagen about 10 years ago to set up his own laboratory, we began a collaboration to study two interrelated signaling pathways of the primary cilium: the pathway for cell differentiation in human embryonic stem (ES) cells and a growth factor pathway involved in cell migration and wound healing in fibroblasts. To understand exactly how the primary cilium controls cell migration, which is a facet of embryonic differentiation and wound healing in the adult, we began reexamining one of the first cells in which the primary cilium was described—the fibroblast. We examined mouse embryo fibroblasts from normal animals; as a control, we used fibroblasts from similar animals with a mutation in a protein (IFT88) essential for cilia formation—the same mutation that Rosenbaum’s group had used with kidney cells.
When we temporarily halted cell-cycle progression by removing nutrients required for division, the normal fibroblasts grew a primary cilium, but the mutant cells did not. When the primary cilium began to grow, the synthesis of PDGFR
Since we had shown that the PDGF receptor was localized to the cilium, we asked whether signaling molecules crucial for ES cell differentiation might also originate at the cilium. The fancifully named “sonic hedgehog” signaling pathway is particularly important in the development of the spinal cord and the brain. Kathryn Anderson’s group at Memorial Sloan Kettering had shown that hedgehog signaling originates in the primary cilium in developing neurons—cell types that also migrate as they differentiate. Our group was the first to show that not only do ES cells contain cilia, but that hedgehog signaling via the patched receptor originates at the cilia—initiating signals that are involved in the normal development of the brain and spinal cord.5
We are just at the beginning of understanding the relationship of the primary cilium to spatial gradients and directions, such as formation of the leading edge in cell migration. We now know that directional cell migration is important in heart formation, which requires signaling from the cilium to initiate the cell migration of cardiomyocytes, resulting in appropriate left–right symmetry determination. And we expect there will be more to come.6
Not surprisingly, the results from a specific cell type, such as the fibroblast or ES cell, may not be general, so it may take a bit longer to provide the explanations needed to show how each ciliary antenna with potentially multiple signaling systems within it works in detail. For instance, we still do not know why and how some specific signaling proteins are localized to the cilium, whereas others are excluded.
As the first organelle ever observed by scientists, it’s interesting to reflect on how the cilium became a necessary component of most cells in the body. Many people now conclude that the complexity of the nucleated cell arose by a series of invasive/symbiotic events. The major organelles—mitochondria, chloroplast, perhaps even the nucleus itself—are the results of such invasions or engulfments within a basic bacterial cytoplasm. My colleagues and I have proposed that the sensory 9+0 cilium could have originated in this way, when a large enveloped RNA-containing virus whose core was the primitive centriole failed to exocytose completely after the invasion of the cytoplasm, leaving a bud.7 When the protein transport mechanism permitted the bud to grow and to accumulate specific membrane proteins, the sensory cilium was born. Later, motility and an efficient coupling between sensory information and motile response evolved.
The importance of the motile cilium should not be overlooked, especially in survival of single-celled organisms, but we now know—what I failed to appreciate at the start of my career—that with or without motility the sensory ciliary antenna had an important role to play in animal evolution, a role that has recently provided us with an increased understanding of how the basic cell biology of the cilium relates to human genetic disease. Because so many of the ciliopathies are genetic diseases, when we understand the signaling systems involved more completely, there is a good chance that gene therapy or small-molecule intervention will be able to used to repair the defects, for example, to grow the primary cilia in mutant cells to cure the disease or delay its onset.
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Peter Satir is a professor of anatomy and structural biology at Albert Einstein College of Medicine.