A New Controller for Thomas the Tubulin Engine

Shuttling tubulin where it’s needed—TIRF time lapse shows a long-short Chlamy cell in the middle column evening out flagellar growth. Image courtesy of Julie Craft and Karl Lechtreck

Shuttling tubulin where it’s needed—TIRF time lapse shows a long-short Chlamy cell in the middle column evening out flagellar growth. Image courtesy of Julie Craft and Karl Lechtreck

Devotees, past and parental, of Thomas the Tank Engine may recall the differences between English railway language and American railroad lingo. In the UK (and on the Isle of Sodor where Thomas runs), a freight car is a goods truck. In the United States a goods truck is a freight car. In cell physiology, a similar confusion can be found in the study of intraflagellar transport (IFT) in cells with cilia or flagella. The biological setting here is not Sodor but long, narrow protrusions extending from cells: a whip-like flagellar tail (as in sperm), a motile cilium that beats in waves along with its neighbors (as in the airway lining), or a non-motile sensing antenna, the primary cilium (as in kidney cells or the retina). IFT’s job is to move tubulin and other building block proteins of the central axoneme up to the growing end and come back for more. The discovery of IFT in 1993 was a breakthrough and led in 2000 to the first genetic link between defective cilia and a raft of genetic human disorders now called ciliopathies.

But is IFT best visualized as Thomas the Tubulin Engine? IFT terminology traditionally favors trains. The cars (UK translation: trucks) are the strings of IFT protein particles being pulled along microtubule tracks by protein motors. But would a convoy of trucks (UK translation: lorries) be a better metaphor for IFT? Or perhaps IFT is more of a tubulin pump, an elevator, or even a conveyor belt? But whether IFT is best visualized as a train, a truck, or a belt, Karl Lechtreck, professor at University of Georgia (UGA) and ASCB member, says that the key question is which cargoes and how much of them IFT is actually transporting into cilia. Is the amount of cargo transport by IFT regulated or does IFT simply pick-up the proteins as they are provided by the cell? In a new Journal of Cell Biology paper, Lechtreck, his graduate student Julie Craft, and colleagues believe they have evidence that answers part of that question. Cells control how much proteins are moved via IFT into cilia, say Craft et al.

To reach that answer, the UGA researchers supplied a missing link in the IFT evidence chain—they imaged tagged tubulin being transported in vivo. Seeing tubulin undergoing IFT was “kind of expected,” says Lechtreck, because the literature on tubulin transport by IFT rests on strong if indirect proof. Still, says Lechtreck, “This report demonstrates tubulin transport by IFT trains with unprecedented clarity.”

There were practical reasons, says Lechtreck, that until recently it wasn’t possible to image IFT at the molecular level. “We could see IFT trains, but we couldn’t determine whether the trains were loaded or not. Only during the last years fluorescently tagged cargoes became available enabling us to see this.”

The Lechtreck lab succeeded in imaging IFT and its associated cargoes in the biflagellate Chlamydomonas by using TIRF (total internal reflection fluorescence) microscopy, which has a bright but extremely shallow depth of field. Every Chlamy, as the beloved lab model organism is known, has two beautifully long flagella, which are easy to amputate by a brief pH shock. The cells will then regrow two flagella within 1 hour, allowing for the analysis of cargo transport and delivery while cells assemble their cilia. The researchers took advantage of the organism’s affinity for attaching with its flagella directly to the cover slip, thus falling within the extremely thin excitation zone of TIRF microscopy. Craft recalls, “Once we isolated a strain expressing GFP-tubulin it was fairly easy to image. Still, the first time that we saw tagged tubulin swiftly moving along the cilium it was very exciting. Once we started to count the number of transport events it became quickly clear that IFT trains are highly loaded while cilia grow but run near empty once cilia have reached their set length.”

So how do cells know when to load the IFT trains and when not to? Lechtreck explains that until very recently, the dominant hypothesis of ciliary length control has been the “balance point model.” Imagine, says Lechtreck, “You have a depot of building material such as tubulin in the cell body. The IFT trains pick up and deliver their tubulin cargoes to the growing end of the cilium. The longer the cilium, the longer it takes the trains to reach the end and to travel back to the cell to pick-up the next load.” As the cilia grow, the route gets longer and the round-trip time gets longer. He continues, “This slows the rate of delivery and eventually the cilium reaches a balance point where the delivery of tubulin to the distant end is matched by length-independent depolymerization of the cilium.”

The balance point model’s beauty is that it is a minimal model, Lechtreck says, “It demonstrates that it is possible to determine ciliary length simply by using a fixed number of IFT trains traveling at a fixed velocity without the necessity to actually measure ciliary length or regulate IFT loading.” As so often happens, real life is much more complicated. To gain insight into the process by which cells regulate cargo loading onto IFT, Lechtreck and his colleagues passed Chlamydomonas through a syringe to shear off its flagella; this will generate a small subpopulation of cells that has lost only one of its two flagella. While the lost organelle is replaced, cells will possess one long non-growing and one short growing flagellum.

Simultaneous imaging of IFT and tubulin—the major building block of cilia—by TIRF allowed the Lechtreck lab to look “inside” the trains moving up the non-growing cilia. They were running empty. But in the growing cilia, the trains were fully loaded and delivering their tubulin cargo to the growing end. Clearly the load or not-to-load decision was being made separately for each cilium of a cell. “What this shows is that the cells ‘know’ which of its cilia is too short and respond by specifically loading the trains entering the shorter growing cilium,” Lechtreck explains. “When the cilium is long enough, the cell slows the loading so that trucks are arriving at the end with no tubulin to deliver. The cilium stops growing.”

Thus, ciliary size is not simply a function of supply of building materials and the capacity of the transport system. Lechtreck proposes that cells measure the length of their cilia and when cilia are too short the cells respond by increasing the loading on the IFT trains. “We’re suggesting that the cells measure and regulate many steps of ciliary assembly. The next question is how does the cell control how much tubulin is carried by an IFT train? And we don’t know that yet,” Lechtreck said.

Work by other labs implicates several protein kinases in the regulation of ciliary length. Lechtreck suspects that they are involved but how they are tied into measuring ciliary length, relaying length information to the cell body, and the loading of IFT trains is unclear. To get to the molecular mechanism, the Lechtreck lab is switching from train spotting to finding the actual switch tower. They are looking for this controller mechanism down the axonemal tracks along which the IFT trains run, somewhere near the basal body where the cilium is anchored.

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.