IFT cargo and motors associate sequentially with IFT trains to enter cilia

Abstract

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone (TZ). How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors kinesin-II and OSM-3, as well as anterograde cargo proteins IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or TZ function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the TZ.

Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A

“…The idea of SWIM is to excite and photobleach only a small region of the sample (5-15 μm diameter), allowing continuous and long-term entry of “fresh” proteins, which have not yet been photobleached, into the excited region. Using this approach, we observed that BBSome subcomplexes diffuse rapidly in the dendrite (Figure 2A and Video S2), similar to what we have observed before for IFT dynein and kinesin-II (Figure S1A) 11 . From these image sequences, we extracted single-particle trajectories, which were used to calculate the diffusion coefficients using a covariance-based estimator 24 .…”

“…The distribution of static localizations from the ciliary-entry events also showed a sharp peak, slightly shifted towards the dendrite (at ∼0.2 μm ; Figure 3E) but without the long tail into the dendrite. The distribution of the moving localizations of BBSome entry events (α > 1.2; Figure 3E right) looked similar to those we observed before for the IFT motors 11,34 , revealing the characteristic shape of the initial part of the cilium: relatively broad at the ciliary base, tapering at the TZ and “bulging” out at the proximal segment of the cilium. We next looked into the distributions perpendicular to the ciliary long axis.…”

“…We observed that individual BBSome, IFT-B and IFT-A subcomplexes enter the cilium a�er first pausing for a while, apparently being incorporated into immobile, assembling IFT trains at the ciliary base (Figure 7D), similar to what we observed before for IFT-dynein 11 . A recent electron-microscopy study in Chlamydomonas showed that anterograde IFT trains in different stages of assembly lined-up at the ciliary base, tethered at the distal end of the TZ, with IFT-A and IFT-dynein linearly oligomerizing from front to back on an IFT-B scaffold 10 .…”

“…The fluorescence intensity was measured on ImageJ by manually selec�ng a region containing fluorescent signal and a region next to it within the illuminated area, for background correc�on. Numerical simula�ons of single-molecule trajectories: To determine the impact of bleaching on the measured pause �me, entry events were numerically simulated (scheme illustrated in Supplementary Figure 5A), similar to a previous study 11 . Simulated tracks were allowed to dock at a given loca�on along a 1D cilia la�ce, with the docking loca�on randomly picked from the distribu�on of docking loca�ons obtained experimentally.…”

“…How the third subcomplex, the BBSome (complex of 8 BBS proteins) associates with anterograde IFT trains remains unknown. In recent years, FRAP 8,9 and structural 10 studies in Chlamydomonas , as well as a single-molecule fluorescence study in C. elegans 11 , have revealed a structural and dynamic picture of how anterograde IFT trains assemble at the base. It appears that IFT trains tether from one end to the TZ while being assembled, with IFT-B forming a scaffold to which IFT-A and IFT dynein attach subsequently from the dendritic side 10 .…”