The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined.
Primary cilia are tiny protrusions from the cell surface, which have a central role in processing sensory stimuli, such as light or odorants. Cilia are also involved in mediating the response to developmental signaling molecules, including Sonic Hedgehog, and may help to convert mechanical signals into electrical or chemical ones. Primary cilia are made up of an axoneme—a core structure that consists of microtubules extending along the length of the cilium—ensheathed by a membrane that contains a number of receptor proteins.
These receptor proteins travel up and down the cilium, and it is generally assumed that an active process known as intraflagellar transport is responsible for their movement. This process is mediated by motor proteins called kinesins and dyneins, which carry cargo proteins along axonemal microtubules. However, it has been difficult to study the transport of individual receptor proteins directly because they are uniformly distributed over the membranes of the cilia.
Now, Ye et al. have shown that intraflagellar transport is not the most important mode of transport for membrane proteins within primary cilia. By labelling individual receptors with a fluorescent dye and then filming their movements under a microscope, Ye et al. found that the receptors generally did not show the directed, linear motion that would be expected from intraflagellar transport. Instead, much of their movement occurred through passive diffusion, with occasional short bursts of directed motion.
To investigate how rapidly receptor molecules could move through the cilium in this way, Ye et al. used a technique called fluorescence recovery after photobleaching (FRAP). This involves using light to bleach the fluorescent dye attached to receptor molecules in part of the cilium, and then measuring how long it takes for the fluorescence to return as a result of other labelled molecules moving into the bleached area: the shorter this time, the faster the movement of the molecules. It took less than a minute for fluorescence to be restored within a primary cilium, indicating that passive diffusion with occasional active transport can move proteins rapidly through the structure.
By using drugs to inhibit intraflagellar transport, Ye et al. confirmed that the majority of membrane protein transport within primary cilia occurs via diffusion. Further studies are now required to determine whether this is also the case for other molecules that travel along cilia, and whether intraflagellar transport may have a more important role in the assembly of these structures.