Single-Molecule Investigation of Intraflagellar Transport Dynamics at the Flagellar Tip

Abstract

In the past decade, cilia/flagella have come to be known as essential sensory organelles for cells. They perform their sensory duties via signaling pathways comprised of transmembrane signaling proteins (TSP) and intraflagellar transport (IFT) machineries that are responsible for translocating TSPs within the flagellum. While IFT average speed, IFT train frequency, and IFT train size have already been studied, little is known about IFT dynamics at the flagellar tip region where anterograde IFT trains deposit their cargo and rearrange for retrograde transport to the cell body. To address this issue, we use single-molecule fluorescence imaging methods to study the motion of GFP-tagged BBS4, an IFT-associated protein that is part of the BBSome complex, at the flagellar tip in Chlamydomonas reinhardtii. These investigations have yielded the following results: (i) In the tip region, BBSomes remain attached to their cargo TSPs and diffuse along the flagellar membrane with a diffusion coefficient of 1130 nm2/s. This result contrasts with the current understanding that the BBSome either remains bound to IFT train on the microtubule or diffuses in the flagellar lumen. (ii) On average, BBSomes remain at the flagellar tip for 2.6 seconds before undergoing retrograde IFT. (iii) Our BBS4-GFP image-size investigation has helped further uncover the nature of the BBSome's oligomerization on IFT trains both during active transport along the flagellum and during the turn-around period at the flagellar tip.