Single-Molecule Investigation of the Intraflagellar Transport of Membrane Signaling Proteins

Abstract

In the past decade, cilia/flagella have come to be known as essential sensory organelles for cells. These cilia/flagella perform their sensory function via signaling pathways that include flagellar membrane signaling proteins and intraflagellar transport (IFT) machinery comprised of IFT particles and BBSome particles. While the membrane signaling proteins are responsible for collecting information regarding a cell's environment, the IFT machinery has been implicated in effecting the delivery of this information through the translocation and proper positioning of the signaling proteins along the flagellar body. Unfortunately, current understanding of this intraflagellar translocation and positioning is limited to rough measurements of average IFT speeds with little regard for speed fluctuations and the causes thereof. Further, the equally important diffusive motion of signaling proteins in the flagellar membrane has been largely ignored. As such, we use single-molecule fluorescence imaging methods to study the intraflagellar motion of BBS4-GFP, IFT20-GFP, and Pkd2-GFP, a transmembrane signaling protein, in Chlamydomonas reinhardtii. (i) In the flagellar entry region, we have found that Pkd2 largely performs Brownian diffusion, implicating lateral membrane diffusion as a primary flagellar entry mechanism. (ii) In the flagellar body, we have found that IFT trains exhibit speed changes of approximately ± 400 nm/s, which we interpret as events of dropping off or picking up membrane signaling proteins. Futhermore, we have observed the average speed of an IFT train with a BBSome attached to be 300 nm/s slower than the speed of a train without a BBSome, indicating that the BBSome carries at least one membrane signaling protein. (iii) Finally, at the flagellar tip, BBSomes remain bound to their associated membrane signaling proteins, diffusing in the membrane at the tip for an average of two seconds before undergoing retrograde IFT.