Single-Molecule Investigation of the Flagellar Membrane Signaling Protein Entry Mechanism

Abstract

The composition of the flagellar membrane differs from that of the plasma membranes--it is enriched with a unique set of membrane signaling proteins. How these flagellar membrane proteins, which are synthesized in the cytoplasm, enter the flagella across diffusion barriers at the flagellar base has become a significant point of inquiry. Using single-molecule fluorescence imaging methods, we have investigated the intraflagellar transport (IFT) processes of Pkd2-GFP, an important flagellar signaling protein responsible for polycystic kidney disease, and tested two proposed models. In Model I, vesicles carry the proteins to the flagellar base and fuse with the membrane before or after the diffusion barrier to allow the flagellar membrane proteins to enter by lateral diffusion; and, in Model II, the flagellar membrane proteins are bound to the BBSome-IFT-particle machinery (kinesin or dynein motors carry IFT particles, which, in turn, carry BBSomes) at the flagellar base and are carried through the basal body transition fibers into flagella. We have observed Brownian diffusion of Pkd2 at the Chlamydomonas flagellar entry region, suggesting that Model I more accurately describes the entry mechanism. We have further studied the turnaround mechanisms of the IFT machinery at the flagellar tip. Our preliminary results indicate that the IFT machinery does not dissociate, diffuse, and re-associate at the flagellar tip for the return trip to the cell body.