Final Report Summary - DISSECTIFT (In vitro reconstitution and mechanistic dissection of Intraflagellar Transport in C.elegans sensory cilia)
Intraflagellar transport (IFT) is a universally conserved transport that is essential for the assembly of nearly all flagella and cilia throughout the eukaryotic phyla. While the central importance of IFT for ciliary construction and function is abundantly clear, we still lack a detailed molecular understanding of this highly specialized transport process. Indeed, IFT differs considerably from the cytosolic transport on microtubules, provoking the question whether the cilia-specific kinesin-2 and dynein-2 motors are evolutionary adapted for ciliary transport. In our work, we provided detailed molecular explanations for several fundamental questions that persisted in the field for decades. To begin with, what is the mechanism of specific motor recruitment and activation for IFT? How do several kinesin-2 and dynein-2 motors on the IFT trains coordinate their actions for efficient transport and how do they ensure a collision-free transport on the same microtubule doublet? We embarked on an ambitious bottom-up approach to identify the minimum requirements to reconstitute a given function in a cell free system. The IFT machinery can be regarded as an intricate molecular puzzle with ~25 different core subunits that form the multimega Dolton IFT trains to which the oppositely directed kinesin-2 and dynein-2 motors are recruited for bi-directional transport in the cilium. To assemble a transport competent IFT-motor complex from these subunits, we used purified components of the IFT machinery of C. elegans. Our functional reconstitution studies identified the DYF-1 protein of the so-called IFT-B sub-complex as a key subunit to incorporate the OSM-3 kinesin-2 motor into the tetrameric OSM-5/OSM-6/DYF-6/DYF-1 complex. This DYF-1-mediated incorporation was sufficient to fully activate the auto-inhibited OSM-3 for directional transport. Our functional reconstitution studies thus delineated for the first time the molecular mechanism of specific motor recruitment and activation for efficient IFT. In the absence of dyf-1 function in vivo, the OSM-3 motor indeed fails to dock onto the IFT train and solely displays diffusion in the cilium, a phenotype that is now explained by our reconstitution approach. This reconstituted IFT-kinesin-2 complex in fact represents the first physiologically relevant kinesin-cargo complex in general and thus opens the door to understand the mechanism of kinesin activation through binding to its designated cargo. Another key feature of IFT is the near 2-dimensional transport geometry where the ciliary motors are lined up on after the other to move the IFT trains, provoking the question of efficient motor cooperation. This specialized geometry can be approximated well in vitro by lining up motors on a DNA-scaffold. By doing so, we provided a mechanistic explanation for how kinesin-2 motors stay out of each other’s way during co-transport. Our simulations suggest that this is achieved simply by tuning the reattachment rates of the motors to the microtubules. The latter is true for uni- and multi-cellular model organisms suggesting that this may be general feature to adapt kinesin-2 motors to ciliary transport. We unmasked another adaptation of the kinesin-2 motor that would allow a collision-free bi-directional transport on the axonemal microtubule doublets in vivo. The kinesin-2 and dynein-2 driven IFT trains were shown to use the same microtubule doublet of the axoneme. The latter provokes the intriguing question of how head-on collisions are prevented during the two-way traffic in such spatially restricted compartment. We demonstrated that the kinesin-2 motor has a built-in property to take left-handed steps that eventually restricts the motor to one side of the doublet, clearing the way for the on-coming traffic. The kinetic adaptations we uncovered in our reconstitution studies collectively support the notion that the kinesin-2 motor has co-evolved with the ciliary machinery to specifically work on axonemes. Many fundamental questions in the field still await clarification at the molecular level. How do small protein subunits assemble into gigantic IFT trains and how are these trains remodelled to reciprocally activate the kinesin-2 and dynein-2 motors? The experimental platform that we established now sets the stage for the mechanistic dissection of the convoluted IFT process in functional reconstitution assays.