It has been a now-you-see-it, now-you-don’t piece of the ciliary apparatus. During cell division, the transition zone (TZ) between the microtubules of the axoneme (the cilia’s scaffold) and its anchoring basal body vanishes, only to reappear when a new cilium is assembled. Researchers suspect that the TZ is the regulatory gateway for cilia, both motile and primary, where genetic defects have been linked to a long line of human diseases including retinal degeneration and kidney disease. Now using a quirk in cell division in the unicellular Chlamydomonas, whose flagellum is structurally identical to that of eukaryotic motile cilia and flagella, researchers at Yale have isolated the elusive TZ and analyzed its proteome. The Yale researchers have identified 10 genes in the transition zone of Chlamy (as the world-renowned lab model organism is known) with human gene analogs that have been implicated in these “ciliopathies.”
The study’s authors, ASCB members Dennis Diener, Pietro Lupetti, and Joel Rosenbaum, also report in Current Biology1 finding a group of six ESCRT (endosomal sorting complexes required for transport) proteins in the TZ proteome. In a phone interview, Diener and Rosenbaum said this was an intriguing discovery as ESCRT proteins are widely deployed elsewhere in cells for pinching off vesicles and membranes, common routes for extracellular signaling. This suggests to Diener and Rosenbaum a mechanism for cilia to act as broadcasting stations for cell-cell signals rather than passive antennae. (Writing with Christopher Wood of Harvard Medical School, Rosenbaum extends this concept in a new Trends in Cell Biology review paper.)
Viewed from above, the Chlamy TZ is a nine-pointed, star-like structure surrounding a central cylinder, with Y-shaped connectors holding the microtubule doublets that make up the outer ring of the axoneme in place against the ciliary membrane. The exact structure of the TZ in Chlamy—its star-like cross-section and the central cylinder—is not found in vertebrates but TZs with Y-connectors and many of the same ciliary proteins are common to all animals with cilia including humans.
During Chlamy cell division, the TZ seems to disappear as the microtubules in the flagellum depolymerize and the axoneme shrinks to a stub. Rosenbaum likens the TZ during cell division to the stub end of a salami. “It’s like you’ve taken a salami and shortened it by 90% …and thrown the flagellum away. Underneath the end that’s left, you cut off a piece. That’s the transition zone. It’s the little bit of the flagellum that’s left between the cilium itself and the centriole basal body.”
Isolating the TZ salami stub has not been easy, said Diener. Hints emerged in recent work from Chlamy studies by Lynne Quarmby at Simon Fraser University that a TZ stub remained intact following flagellar resorption prior to cell division. It wasn’t until he and Quarmby collaborated that Diener spotted the TZ stubs trapped within the flagellar collar, a proteinaceous sleeve that serves as a passage for the flagellum through the cell wall. “When I saw them in the electron microscope (EM), I said, ‘This is what we’re looking for.’ Before, we’d talk about the TZ but there was no way to isolate it. Now we find the cell isolates it for us.”
That this shrunken bag containing the TZ remains caught in the flagellar collar may be a phenomenon peculiar to Chlamy, but, said Rosenbaum, “Dennis took advantage of that. He took cells in which the flagellum had been deleted and the transition zones had also been cut off but they got stuck in this accessory to the cell wall.” Once he released the TZs from the collar he was able to purify them from the rest of the cell. That’s a perfect example of researchers using the biology of Chlamydomonas for their own purposes, says Rosenbaum.
The structure of the cilium with its nine microtubule doublets was one of the earliest discoveries in the modern era of EM-driven cell biology. But it was the discovery in the late 1990s of the role of primary or non-motile cilia in disease that started the ciliopathy bandwagon rolling. The identification of intraflagellar transport (IFT) and the motor genes in Chlamy that drive it in both directions provided some of the first disease gene hook-ups of primary cilia. Work by Maureen Barr and by Doug Cole further pointed to the cilium as one of the cell’s repositories of the polycystic kidney disease (PKD) gene products. The ciliopathy bandwagon became a speeding locomotive with work by the labs of Rosenbaum, Greg Pazour, George Witman, and Bradley Yoder showing that the PKD gene products indeed resided on the primary cilia of mouse kidney tubule cells. Knocking out IFT in mouse mutants resulted in the lack of these cilia and the resultant PKD disease state.
It had been known that before cell division, the flagella of Chlamy resorb down to the point where the microtubular axoneme connects to the top part of the TZ. After the flagellum has been lost, the TZ is sealed off at its top by a portion of the flagellar membrane, encasing the TZ. We still do not know why the whole axoneme and TZ are not initially clipped off together, but they are not.
But why hadn’t the encased TZ been isolated before? From his study of the literature, Diener believes that the TZ’s hiding place during cell division had probably been observed 40 years ago by investigators studying cell wall assembly. But they didn’t know what the vesicle was and weren’t interested in cilia, says Diener. “It’s only been in the last decade that the TZ has been recognized as such an important part of the cilium.”
The identification of ESCRT proteins in the TZ was a pleasant surprise, says Rosenbaum. “Up until now, most of the work that has been done on cilia is as an antenna receiving signals. New work from Maureen Barr and our lab says that it’s also sending signals out. There are vesicles being pinched off somewhere in the ciliary apparatus so what this says is that the apparatus for sending signals out from the cilium is present in the ciliary membrane.”
“The implication here is that most of these ESCRT proteins are involved in some way in cilium function,” says Diener. The next step in tracing the normal role of ESCRT proteins in the cilium would be to develop mutants or knockdowns to see what phenotypes arise. “It might be that the ESCRT proteins are involved in pinching the cilium off just before [cell division],” Diener says.
It’s an exciting question to pursue, says Rosenbaum. “Parts of the flagellar apparatus—by which I mean the basal body, transition zone, and the flagellum or cilium proper—have been the subject of a huge amount of study in the last decade because many of the disease-causing products are localized there.” These gene products are associated with some very specific diseases including polycystic kidney disease. “Many of those disease-related genes make proteins which are localized in the transition zone. Since that part of the organelle has been fingered as controlling the entry and exit of things from the cilium proper, that means a lot of diseases are caused directly by either having the cilium not present or incorrectly assembled or by having the wrong protein on the ciliary membrane,” says Rosenbaum.
The last salami slice has more secrets yet, the researchers believe.
1Diener et al., Proteomic Analysis of Isolated Ciliary Transition Zones Reveals the Presence of ESCRT Proteins, Current Biology (2015)
About the Author:
John Fleischman was the ASCB Senior Science Writer from 2000 to 2016. Best unpaid perk of the job? Working with new grad students and Nobel Prize winners.
