Ultrastructural and immunolocalization studies on the interactions occurring between IntraFlagellar Transport components and the ciliary tip structures during IFT trains turnaround in Chlamydomonas flagella

Cilia and flagella are dynamic organelles of the eukaryotic cell that undergo cycles of assembly/disassembly, in a manner that is coordinated in time with the cell cycle. Cilia are composed by more than 600 peptides and their turnover occurs at the plus distal end of the axoneme; since these organelles lack the machinery required for protein synthesis, a bidirectional transport process known as the IntraFlagellar Transport (IFT) is required to provide flagellar precursors and remove turnover products. IFT is carried on by macromolecular complexes (the IFT particles) which are arranged in polymers (the IFT trains) in the space between the microtubular doublets and the flagellar membrane. IFT trains operate as platforms for cargoes and are moved bidirectionally by specific molecular motors, kinesin-2 as the anterograde motor and dynein-1b as the retrograde motor. Anterograde and retrograde IFT trains possess distinct architectures but, up to now, a high-resolution 3D-model is available only for the anterograde trains, while much less is known on the ultrastructure of retrograde trains. At the distal tip of the organelle, the anterograde transport (from the cell body up to the ciliary distal end or tip) is converted into the retrograde transport (from the tip back to the cell body). Such a turnaround process is strictly required for the correct functioning of the IFT process. So far, however, very little is known about the morpho-functional organization of the tip district, where IFT turnaround takes place. In particular, nothing is known on the interactions that might occur between IFT proteins and the distal tip structures. This doctoral work has been aimed at contributing new information for the comprehension of the IFT turnaround process in the model organism Chlamydomonas reinhardtii. We started our studies from the observation that thin sections of flat-embedded flagella often show anterograde IFT trains that contact the distal end of the central pair microtubules (CP), suggesting the direct involvement of CP capping structures (terminal plates and the ring above) in the IFT turnaround process. We confirmed the interaction of anterograde trains with the CP distal end by electron-tomographic reconstruction of flat-embedded flagellar tips. This approach revealed that anterograde trains split into three components after having reached the end of the A tubule, with the outer part of the train that remains associated with the membrane, the inner part, closer to the microtubule surface, that continues its travel and bends to contact the CP plates, and an intermediate part that stops before reaching the tip. The latter region was interpreted as the part of the train consisting of inactive dynein-1b, which is known to dissociate from the anterograde train before its activation and recruitment for the retrograde transport. Then, we sought to obtain further information on the ultrastructural organization of the distal CP segment. We were able to identify a ladder-like structure (LLS) which is distinctive of this region, is intercalated between the two CP tubules, and is resistant to the cold treatments used to depolymerize tubulin. In order to confirm the association between IFT anterograde trains and the capping CP structures, whole cells were treated with inhibitors of Ca++-dependent protein kinases before flagellar demembranation and negative staining. These inhibitors block the release of kinesin-2 from the anterograde trains and, consequently, IFT turnaround at the tip. As expected, we observed a massive accumulation of IFT particles around the CP terminus. Successively, we analyzed by immunoelectronmicroscopy the specific distribution of the three protein complexes present within the IFT particles. At this purpose, we carried out a series of immunolabeling experiments on grid-absorbed demembranated cells or on sections of resin-embedded samples, using antibodies directed against subunits of the IFT-A complex (IFT139), and of the two IFT-B subcomplexes IFT-B1 (IFT74 and IFT81) and IFT-B2 (IFT172 and IFT57). Our findings suggest that at the tip the IFT-A complex is closely associated with the membrane. On the contrary, both IFT-B1 and IFT-B2 antibodies labelled the distal CP region, though, interestingly, with distinct spatial distributions. IFT-B2 labeling was restricted to approximately the distal 200 nm-segment of the CP, which contains the LLS, and gold particles were never found more distally, above the terminal plates, while IFT-B1 labeling extend also to the ring. The whole set of immunoelectronmicroscopy data indicates that the IFT-B1 and IFT-B2 subcomplexes differentially interact with the distal CP region and its capping structures, and suggests that the IFT-B1 subcomplex might be a main component of the CP capping structures. Accordingly, in our negatively stained samples the cap was shown to consist of thin elongated elements, frequently with a sort of small knob at their mid region; these elements fit quite well with the available IFT-B 3D model. The possibility that IFT-B1 proteins are involved in the formation of the CP cap was confirmed by the analysis of a series Chlamydomonas mutants with defective IFT, which related the presence of the CP cap to the establishment of a fully cycling IFT process. Our data sustain a model of IFT turnaround in which i) the IFT-A complex turns around quickly, remaining associated with the membrane, ii) IFT-B1 and IFT-B2 follow a more complex pathway, during which they separate and differentially interact with the CP distal segment, iii) IFT-B1 directly contribute to the formation of the CP cap. The LLS component, which is ectopically assembled also in mutant strains devoid of the CP tubules, is likely to act as an anchoring structure for IFT-B2 during IFT turnaround.