Dissecting The Function And Mechanism Of IFT54 In Cilia Assembly

Cilia or flagella are microtubule-based hair-like organelles located at the surface of most eukaryotic cells.They play crucial roles in cell motility,sensory reception,signaling transduction,embryo development and cell cycle progression.Cilia dysfunction is implicated in a series of human diseases known as ciliopathies,including renal anomalies,polydactyly,situs inversus,chronic bronchitis,etc.Multiple studies have shown that some ciliopathies are associated with mutations in intraflagellar transport(IFT)related genes.Therefore,the research on IFT will provide new insights into the diagnosis and treatment of ciliopathies.IFT particles are composed of three parts: IFT complexes made up by IFT-A and IFT-B subcomplexes,anterograde transport motors Kinesin-2,and retrograde transport motors IFT dynein.IFT ferries cargoes bidirectionally between the ciliary base and tip,and is required for cilia assembly,disassembly,maintenance and signaling transduction.Even though the common model of IFT process has been established,the specific role of individual IFT subunit is still unclear,and the mechanism of interaction and dissociation between IFT complexes and motors remains to be explored.In this thesis,we choose the IFT-B subunit IFT54,which was reported as the pathogenic gene of several ciliopathies,to do further research.By insertional mutagenesis,we obtained an ift54 mutant in Chlamydomonas which completely blocks flagella biogenesis beyond the transition zone.Loss of IFT54 leads to instability and degradation of IFT20,but does not affect the assembly and localization of IFT-B complex.Although IFT54 colocalizes with mitotic spindle and microtubule bundles in the division furrow during mitosis,ift54 mutant cells are able to form proper spindle and undergo apparently normal cell division.The N-terminal calponin homology(CH)domain of IFT54 is not required for the tubulin transport and ciliogenesis,but influences the binding with axoneme and the amount of IFT54 proteins entering flagella.The C-terminal coiled-coil(CC)domain is involved in the interaction with IFT20 and thus stabilizes IFT20,which is essential for the incorporation of IFT54 into IFT-B complex and the recruitment of IFT54 to basal body.Expressing CC domain in ift54 mutant cells restored the cellular level of IFT20,yet only partially rescued the aflagella phenotype with short swollen flagella accumulated IFT proteins,indicating that the protein sequence between CH and CC domain of IFT54 has a unique function in ciliogenesis independent of IFT20.Further research demonstrates that the residues 261-275 of IFT54 mediates the process of IFT dynein loading to IFT-B complex and entering flagella,while the residues 342-356 of IFT54 regulates the binding strength of IFT-B complex with Kinesin-II,thus inhibits the motor activity of Kinesin-II.To sum up,we have carried out detailed studies on the functional mechanism of IFT54 in flagella assembly,and reveal that IFT54 participates in regulating the attachment of motor to IFT-B complex for the first time.Our results not only provide a key supplement to the understanding of IFT54,but also promote the further refinement of the intraflagella transport model.