Cilia Tricks: From Cell Biology to Ciliopathies

In Latin, “cilia” means eyelashes, and anyone who has seen these tail-like projections on eukaryotic cells can see why. Extending off the cell membrane, cilia contain a microtubule array known as the axoneme, a characteristic pattern of nine sets of double microtubules arranged in a ring around a pair of single microtubules. Some primary functions of cilia are movement and sensory reception.

In 1965, when Professor Joel Rosenbaum of the Molecular, Cellular and Developmental Biology department embarked on his first key research project, few were aware of cilia’s importance. Yet over the past four decades, Rosenbaum’s research has shed light on the role these tiny organelles play in deadly human diseases known as ciliopathies. In recent years, his research has gone well beyond basic cell biology and led to breakthroughs in medicine as well.

Rosenbaum was initially interested in the assembly of the different organelles in the cell, including mitochondria, chloroplasts and ribosomes. The complicated formations of organelles were especially compelling for him. His original work involved mitochondria, back when researchers first started to discover that mitochondria had the ability to synthesize protein. However, wanting to study an organelle that could be easily visualized, Rosenbaum switched his focus to cilia and flagella. Since he could easily cut off these appendages, it was possible for him to study the mechanism and kinetics of reassembly.

The First Experiment: Flagellum Regrowth

Rosenbaum conducted his first important experiments on cilia and flagella from 1965 to 1970, beginning at the University of Chicago and finishing at Yale. Using Chlamydomonas, a haploid biflagellate alga with the genetic characteristics of yeast but the cell architecture of multicellular organisms, Rosenbaum cut off the flagellum of the Chlamydomonas and examined its regrowth. To visualize how the structure re-formed, Rosenbaum and his team allowed the flagellum to grow halfway back before adding radioactive amino acids to track the rest.

The results were quite surprising: it took only about an hour for the flagellum to grow back. As radiation was only detected around the tip of the flagellum, Rosenbaum was able to conclude that the flagellum itself was not able to synthesize protein. This suggested that proteins for the flagellum were all made in the cytoplasm and then transported to the tip of the flagellum.

Rosenbaum reflected, “It was nice to see, but very perplexing because the cilia and flagella contain about 350 polypeptides. How does it all come together so rapidly to allow the assembly of this rather complicated organelle? How does the cell know that you’ve cut its flagellum off and turn on new mRNA and protein synthesis in a massive sort of way to build a new flagellum? Finally, once the flagellum reaches its full length [10 to 12 microns long], how does it know to stop? What limits its growth?”

Elucidating the Protein-Transporting “Elevator”

Armed with these questions, Rosenbaum embarked on a thirteen-year investigation at Yale from 1967 to 1990 that focused primarily on the basic cell biology of organelle synthesis and protein assembly. Beginning with the question of how flagellum regrowth is initiated, Rosenbaum cut off the flagella of Chlamydomonas once again and used inhibitors of RNA followed by Northern blots. This was done to detect increases of specific flagellar RNA synthesis after the detachment of flagella. When he used colchicine, a natural product that allows protein synthesis but inhibits microtubular assembly, he observed that flagellar proteins were indeed still created even though assembly did not occur.

These results raised several questions. What drove the protein transport? And what was the mechanism?

Keith Kozminski, a graduate student in Rosenbaum’s laboratory, decided to investigate the “elevator” that transported such proteins from the cell body to the tip of the flagellum. In 1977, he performed an experiment in which he placed tiny Latex beads in the fluid around the cells. He noticed that some beads attached to the outer surface of the flagellum and moved up and down the length of the organelle. To further investigate such movement, Paul Forscher, an expert on optical imaging, joined the group and used new high-resolution microscopy to watch particle movement along the length of the flagella, which was then named intraflagellar transport (IFT).

To further understand IFT, Rosenbaum conducted a series of experiments that exploited many uniquely useful properties of the Chlamydomonas. One of these properties is the touching and adhering of the flagella of two organisms during mating. Gamete activation follows, leading to the loss of the cell wall and the formation of the dikaryon (pair of unfused haploid nuclei). The flagella are then absorbed into the cell to form the zygote.

Rosenbaum mated a normal Chlamydomonas with a mutant that could not fully grow its flagellum. Fluorescent labeling revealed that upon gamete fusion, full growth of the flagellum was added onto the tip of the mutant zygote (see Figure 3). This suggested the need of a motility system for over 250 polypeptides to be added onto the tip.

With differential interference contrast (DIC) and transmission electron microscopy (TEM), the IFT particles were detected as bulges along the flagellum in between the outer microtubules and the membrane. Kinesin II moved from the base to the tip at about 2.5 microns per second, whereas cytoplasmic dynein 1b moved from the tip to the base at 4 microns per second.

Rosenbaum also discovered temperature-sensitive genetic mutants of kinesin that stopped IFT when the temperature was raised by 10oC. After raising the temperature of these cells, Rosenabuam found that 16 to 20 polypeptides and motors were missing. Surprisingly, most of these proteins were cell body proteins, not flagellate structures.

The Ciliary Hypothesis of Polycystic Kidney Disease

Advances in genomics allowed for comparisons between Chlamydomonas genes and those of other organisms. To Rosenbaum’s surprise, IFT-88 came up in the database as a homolog to the mouse protein Tg737, a protein implicated in polycystic kidney disease (PKD), a genetic disorder characterized by the growth of cysts that affect normal kidney function.

The relationship between Chlamydomonas and PKD-affected mice was not at all obvious, as these two organisms are separated by thousands of years of evolution. To investigate this matter, Rosenbaum applied his knowledge of IFT-88’s effect on Chlamydomonas to knock out mice. Rosenbaum knew that if IFT-88 were knocked out in Chlamydomonas, then flagella would not form.

When tg737 was knocked out in mice, the cilia of kidney tubules were reduced or absent, which caused the “ciliary basis of polycystic kidney disease.” As in earlier experiments with Chlamydomonas, the absence of cilia in mouse kidneys stimulated cell division, resulting in the formation of fluid-filled cysts in the kidney.

Rosenbaum recollected how his ciliary hypothesis of PKD was not initially well received. However, in the following decades, many diseases were traced back to ciliary defects.

Beyond the Kidney: Cilia’s Role in Other Diseases

Retinal degeneration is another important field for ciliary research, as the outer segments of vertebrate photoreceptor rods and cones are formed from cilia. The transport of proteins and lipids from the cell to the tip of the cilia is critical for the functionality of the cilia, since about 10% of the tip degenerates each day. Ciliary defects can lead to retinitis pigmentosa and blindness.

Jean Bennett, a former undergraduate at Yale and now a professor at the University of Pennsylvania School of Medicine, was a pioneer in using gene therapy to restore sight. She found that the injection of the genes that affect movement up and down the retinal cilia led to the restoration of sight in dogs and, very recently, in humans as well.

PKD and retinal degeneration are not the only diseases associated with ciliary defects. Other ciliopathies include Bardet-Biedl syndrome (a disorder characterized by obesity, mental retardation, and renal failure), Meckel syndrome (a rare and lethal disorder characterized by central nervous system malformations and various developmental defects), situs inversus (a congenital condition in which internal organs like the heart are reversed), and primary ciliary dyskinesia, i.e. PCD, (a rare genetic disorder involving defects in the action of the cilia lining the respiratory tract and fallopian tubes).

Finally, Rosenbaum is also pondering whether cancer may be the result of cilia loss. As cilia must be absorbed during cell division, the idea is that its absence may lead to the uncontrolled cell division that leads to tumor formation.

Future of Drug Development

When studying Chlamydomonas, Rosenbaum astutely took advantage of the detectable green from the chloroplasts to visualize the effects of certain chemical compounds on the PKD gene. The first response when two flagella touch during mating is an influx of calcium ions into the channels and a consequent loss of the cell wall. Without the cell wall, the addition of detergent lyses the chloroplasts, turning the surrounding medium green.

This provides a quick way of seeing whether the PKD channels are functioning or not. Basically, if the channels are not functioning and the flagella are touching, then no signal will go down the flagella, the cell walls will not come off, and upon addition of detergent, the medium will not turn green.

Over 25,000 different chemical compounds have been screened in the Yale Center for High Throughput Cell Biology. So far, six have allowed the green color to emerge when defective PKD channeled-cells mate. From these compounds, effective drugs can be found to correct for human PKD mutations. The next step is applying this to experimental mouse models.

When he first began researching cilia, Rosenbaum had not foreseen the vast assortment of applications of ciliary research. Yet in the past few decades, he has paved the way for many biological and medical innovations. Rosenbaum and his colleagues are slowly teaching the medical world about the mechanics of ciliopathies and providing researchers with the tools they need for potential treatments.