primary cilium pushed its way into cell biology's center ring with the discovery that this "irrelevant" vestigial organelle was connected to a common and fatal human disorder, polycystic kidney disease (PKD). In the years since, a long list of diseases and disorders have been classified as ciliopathies while the primary cilium currently has 2,347 citations on PubMed.It's been nearly 14 years since the
Yet basic—and unexpected—discoveries about the primary cilium are still being made. In the latest issue of Molecular Biology of the Cell, Chelsey Chandler Ruppersburg and H. Criss Hartzell of Emory University report that ANO1 (also known as Tmem16A), a gene encoding a calcium-activated chloride channel (CaCC) known to be involved in epithelial fluid secretion, has a unsuspected secret life early in the interphase of the cell cycle. ANO1 creates a ring-like structure at the apical membrane that the researchers term the "nimbus." It seems to be part of a scaffold for assembling the proteins necessary for ciliogenesis before the docking of the mother centriole that provides the basal body on which the primary cilium develops. Blocking ANO1 with drugs or knocking the gene down with short hairpin RNA (shRNA) seriously interferes with ciliogenesis. With limited or no ANO1, cultured epithelial cells fail to form primary cilia or produce shorter cilia.
It was the nimbus that first caught Hartzell's attention and it was ANO1 that led him there. "I've been working on ion channels pretty much all my career," says Hartzell, but only in the last five years has anything been known about the genes encoding CaCCs. The ANOs (anoctamins) are a scientifically fractious family of 10 genes, two of which, ANO1 and ANO2, are anion (chloride) channels. The functions of the other eight are unknown or contentious. Some ANOs seem to be phospholipid scrambling enzymes, known as phospholipid scramblases. How could the two subgroups of anoctamins share sequences and domain structures and yet perform such different actions? Hartzell, who thinks that ion channel genes might play multiple roles at different times in a cell, wanted to see where he might find ANO1 at work.
"I believe really strongly in letting your data lead you in your research. I was just doing immuno-staining of kidney cortical collecting duct cells," Hartzell explains. He wanted to track ANO1 as cells polarized into an epithelium, expecting ANO1 to be located on the apical membrane. "It was almost an experiment that I didn't do because I was so confident of the outcome. Then I saw these crazy rings."
Ruppersburg had just joined the lab as a grad student, Hartzell recalls. "I said to her, 'Why don't you figure out what these weird rings are?'" Two years later, Ruppersburg can report, "We didn't know where it was going to lead, but it just happened that all roads led to the cilium."
Call them rings, nimbi, or "compartments," similar structures had been observed in the literature before, but their punch-line remained vague. Cultured under conditions that "disfavored" quick progression to ciliary formation, cells expressing ANO1 developed sharply defined rings, usually one to a cell, made up of distinct puncta, at the apical membrane. By varying culture conditions, closely tracking labeled ANO1, and analyzing the proteins assembling around the nimbus, the order of the ring became clear, says Ruppersburg. "This nimbus forms prior to any practical detection of the primary cilium. It's collecting these proteins that we know are important for cilium formation."
Plotting the spread and then concentration of ANO1 on the apical membrane in temporal and spatial terms was easy, she says. "The pieces came together naturally through a lot of help from collaborators who were really familiar with cilium research." But she adds, "The hard part for us is figuring out what this chloride channel is doing in the cilium."
Their data strongly support their model that the ANO1 nimbus is a scaffold for the next step in nucleating and then building the cilium. But Hartzell is unwilling to jump ahead of these results. How does ANO1 actually work? Does it change the membrane's electrical potential, alter its hydrostatic pressure, or does chloride directly regulate other proteins? Hartzell is reluctant to jump to any conclusions about what a CaCC is doing in ciliogenesis, although he says that over a beer, he might be willing to speculate.
As a chloride ion channel person, Hartzell says, "It's always fun to come into a new field as an outsider because you don't know any of the inside stories. As an outsider, you're unbiased." You're also cautious for fear that you might be missing something everybody in the field already knows, he says.
So what have they added to the primary cilium story? Says Ruppersburg, "My impression from the literature is that there are steps in ciliogenesis that happen in a certain order that the majority of researchers agree on. It's when you get into the details and the transition between those steps that it seems there are huge gaps and missing information."
Has the ANO1 story filled any of those gaps? "I'd say we added another question," says Hartzell. "I think we need to figure out exactly what role ANO1 is playing. We have some data that ANO1 is playing a role here (in ciliogenesis) but does that mean it has to do with chloride transport or does ANO1 have other functions not related to ion channel function?"
The primary cilium literature awaits the next chapter.
Ruppersburg CC, & Hartzell HC (2014). The Ca2+-activated Cl- channel ANO1/TMEM16A regulates primary ciliogenesis. Molecular Biology of the Cell PMID: 24694595
This work was supported by NIH grants GM60448 and EY11482.