Files
Abstract
Cilia are cellular extensions composed from over a thousand distinct proteins. The composition of the cilium is regulated and many of the functions of the organelle depend upon a precise protein content. During assembly, a vast amount of proteins must be transported from the cell body to the site of assembly within the cilium. Small proteins enter cilia by diffusion and intraflagellar transport (IFT), the predominant protein transport system in cilia. This applies to tubulin, the main structural protein of cilia. How individual proteins utilize these two delivery modes as well as their respective contributions in ensuring the proper protein environment of the cilium remains unknown. To investigate the roles of diffusion and IFT in ciliary protein transport we utilized single molecule in vivo imaging of fluorescently tagged ciliary proteins. We showed that the microtubule end-binding protein, EB1, localizes to the tips of cilia and is continuously exchanged between the cell body and cilia in an IFT-independent manner. EB1 moves swiftly along the ciliary shaft but displays a markedly reduced mobility near the ciliary tip. These two distinct mobilities were sufficient to accumulate a protein at the ciliary tip in simulations. In contrast to EB1, tubulin enters cilia by diffusion and IFT. The roles of diffusion and IFT in providing tubulin for axonemal elongation were determined using modified tubulin constructs and an altered IFT system. IFT transport was reduced by ~90% when the E-hook was removed from -tubulin; transport was essentially abolished in conjunction with an altered IFT81 protein that cannot bind tubulin. Despite the extreme reduction in IFT, presence of E-hook deficient -tubulin in the axoneme was marginally reduced indicating that the lions share of axonemal tubulin enters cilia via diffusion. We propose that the observed modes of transport utilized by proteins is a reflection of the subcellular distribution of said proteins. Proteins which are equally distributed between the cytoplasm and cilia utilize diffusion as a means to translocate between the two compartments. IFT, on the other hand, is employed to overcome concentration gradients to ensure high quantities of a protein near the ciliary tip to promote ciliary elongation.