Cilia elongation and homeostasis require the transport of building blocks and signalling molecules between the ciliary base and tip through a process called intraflagellar transport (IFT). IFT is driven by multiple copies of the ciliary motor proteins kinesin-2 and dynein-2 attached to train-like assemblies of IFT-A and IFT-B protein subcomplexes. Kinesin-2 transports IFT-trains along a microtubule-based ciliary cytoskeleton towards the ciliary tip during anterograde IFT. The ultrastructure of in situ anterograde IFT-trains was determined by cryo-electron tomography (cryo-ET) in the Pigino lab, but high-resolution structural information of the interactions within and between the IFT-A and IFT-B subcomplexes remains absent. I set out to purify IFT-complexes from the motile cilia of the protist Tetrahymena for structural analysis using negative-stain and cryo-electron microscopy (cryo-EM). I identified Bmh 14-3-3 as a potential novel component of the purified IFT-B complex and found IFT43, IFT25 and IFT56 are likely lost in Tetrahymena IFT. Negative stain EM averages of IFT-A complexes partially fit in the in situ IFT structure, but the complex disintegrated during cryo-EM sample preparation with all tested conditions. This report of purification and sample-preparation conditions contributes to the future optimisation of the IFT-complexes for cryo-EM.

Anterograde IFT-trains reorganize into dynein-2 mediated retrograde trains during IFT-turnaround. IFT-turnaround takes place at the ciliary tip, close to but independent of ciliary microtubule capping structures. The role of the ciliary capping structures remains ambiguous and high-resolution information is lacking. Moreover, high-resolution in situ observations of IFT in mammalian motile cilia and the process of IFT turnaround are missing. I used cryo-ET on motile cilia from porcine trachea to survey IFT and ciliary cap structures in the ciliary tip. I observed ciliary cap structures consisting of two stacked amorphous plates and identified dynein densities in the distal-most plate. This suggests porcine ciliary cap structures consist of a single plate and IFT-turnaround densities, in contrast to five stacked plates previously observed in rodent trachea. I identified retrograde IFT-trains, and putative anterograde IFT-trains with high-contrast membrane-associated densities. A larger dataset of porcine tracheal cilia combined with subtomogram averaging could further the initial observations of novel membrane-associated densities, IFT-turnaround and the ciliary caps.

In interphase cells, centriole-pairs are connected by striated fibrils composed of the coiled-coil protein rootletin along with CEP68, CCDC102B and potentially other proteins. Upon ciliogenesis, the centriole-pairs template the ciliary cytoskeleton and the fibrils merge into a striated fibre called the ciliary rootlet. The rootlet is highly conserved and critical for ciliary functioning and maintenance through an unknown mechanism. The rootlet’s structural organisation, direct protein-protein interactions and striation composition remain unknown. I set out to purify recombinant and native rootlets for analysis with EM and cryo-ET to analyse the rootlet structure and ultrastructure. Tomograms of purified mouse photoreceptor cell rootlets showed a longitudinal periodicity with an amorphous striation and three confined striations each 81.3 nm. Subtomogram averaging showed the absence of lateral regularity in both the filaments and confined striations. The confined striations consist of globular densities associated to filaments, frequently found as parallel filament pairs, with no visible difference in filament density on either side of a striation. These findings rule out a previously proposed collagen-like filament organisation. Extensive optimisation of subtomogram alignments, classification and averaging resulted in a dimer of two-helix coiled-coils. Lateral oligomerisation of rootletin coiled-coils was not seen before in situ, but it remains unknown if the structure represents parallel or staggered rootletin contacts. Finally, I set out to establish a system for recombinantly expressed rootlets in SF9 insect cells and mammalian cells. Regardless of the presence of CEP68 or CCDC102B during expression, the purified fibres from insect or mammalian cells did not contain any striations. The previously observed striations are either not an intrinsic part of rootletin and may arise from unidentified rootlet interacting proteins, or the striations only form with factors such as chaperones that are missing in the current expression systems.