Cilia Admit Larger Proteins Than Thought

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A new fluorescence visualization technique reveals that cilia admit much larger molecules than has previously been seen—a finding that could provide new insights into cell signaling systems from what could be an important new research tool.

Often called antennae for their roles in signaling processes, cilia monitor the cell’s exterior environment, translating mechanical and chemical forces into molecular signals so the cell can respond appropriately. In the kidney, cilia monitor the flow of urine. They sense the wavelength of light in the eye, pressure in cartilage, and blood flow in the heart. Defects in the hairlike protrusions have been implicated in disorders ranging from polycystic kidney disease to loss of vision and hearing.

A team led by Takanari Inoue, PhD, assistant professor of cell biology at the Johns Hopkins University School of Medicine, found that molecules as large as 650 kDa can enter the cilia, which is some 10 times larger than was heretofore thought. That size limit means that 90 percent of the proteins in mammalian cells can make their way into cilia and perhaps affect signaling functions.

In their study in Nature Chemical Biology, the team said that the gateway is not a pore of fixed size but a sievelike process, in which small molecules transfer through more quickly than do larger ones. Inoue said that a key point may not simply be which molecules can make their way into the cilia, but what happens to them once they are there. Inoue told Kidney News that one “highly speculative thought” is that “under some disease conditions, primary cilia get clogged at the base, so that the proteins cannot go in and out, so ... they cannot send a signal.”

The only way into a cilium is through the base from which it protrudes from the cell, so several recent studies have looked into how proteins traffic in and out. Last year, a study in the Proceedings of the National Academy of Science USA concluded that photoreceptor primary cilia have no fixed pore that limits the diffusion of soluble proteins, at least up to 80 kDa. By contrast, a study in Nature Cell Biology concluded that the ciliary base does contain a fixed pore that excludes molecules larger than 67 kDa. These researchers termed the barrier a ciliary pore complex, believing it to be analogous to the nuclear pore complex (NPC), but Inoue’s team overcame some limitations of that study and found a way to document that larger molecules could enter the cilia.

The Johns Hopkins team developed a variation on the chemically inducible dimerization technique that is often used to manipulate signaling molecules. In dimerization, two proteins that would otherwise not interact are each induced to attach to different sides of the same enzyme or other dimerization agent and thereby form a ternary complex. This experiment used rapamycin as the dimerizer.

Inoue’s team engineered a molecule with a cyan fluorescent tag targeted to embed itself in the cilia’s membranes. One end anchored itself to the membrane and the other end contained a rapamycin-binding domain. They then introduced into the cytoplasm their particular “proteins of interest,” molecules with yellow fluorescent tags engineered with an end that would attach to the rapamycin. When they introduced rapamycin, they waited to see what would diffuse into the cilia.

They call their technique a chemically induced diffusion trap because the cilia become the equivalent of a roach motel, where the molecules can check in separately but, once bound together, cannot check out. The researchers repeated the experiment with molecules of increasing size and found that everything they tried, up to the largest size their technique would permit—650 kDa, or 7.9 nm—made it into the cilia.

Small molecules entered more quickly than did larger molecules. The researchers performed calculations on the speed at which the various sizes of molecules diffused, and they proposed that the barrier was more like a sieve than a simple pore.

They proposed “two important implications for how ciliary protein composition is regulated.” First, over long time scales, the exclusion of proteins may not be as important as what happens once they are in—the binding interactions with other cilia-resident molecules that lead to the selective retention of proteins. Second, because signaling reactions are rapid, the sievelike barrier’s role in limiting protein entry could play an important regulatory role.

“I think the surprising thing here is that even large soluble proteins seem to be able to get through this kind of barrier,” said Peter C. Harris, PhD, professor of medicine and biochemistry and molecular biology at the Mayo Clinic.

However, Harris did not see an obvious link to polycystic kidney disease because the experiment dealt with soluble proteins rather than membrane proteins: “The proteins that are involved in simple polycystic kidney disease, like polycystin-1 and polycystin-2, and fibrocystin for the recessive form of polycystic kidney disease, are fairly large and all membrane proteins. So they are not going to get into the cilia in the way that is described in this paper.”

Terry Watnick, MD, director of the Baltimore Polycystic Kidney Disease Research and Clinical Core Center, which funded the study with a grant from the National Institute of Diabetes and Digestive and Kidney Diseases, agreed that most work on polycystic disease has involved membrane proteins, but said, “There is an intimate tie between the cilia and cystic kidney disease. There could be unidentified soluble molecules that may be required for ciliary-based signal transduction pathways.”

Watnick was excited by the possibilities the diffusion trap offers for further research: “You could adapt this methodology to bring other signaling compounds to the cilia to perturb the system and see what the downstream effects are. This could be a useful tool for exploring signaling systems in cilia because it allows you to target molecules to cilia, and Inoue has shown that he is able to do that effectively.”