Functional Exploration Of The IFT-A Complex In Intraflagellar Transport And Ciliogenesis

Eukaryotic flagella or cilia(interchangeable terms)are microtubule-based cellular structures projecting from the cell surface.They play pivotal roles in cell motility and signaling.Ciliary defects are associated with a cohort of human diseases and developmental disorders,termed ciliopathies.Ciliary assembly and maintenance requires intraflagellar transport(IFT)that ferries within the cilium.Intraflagellar transport particles or trains are comprised of IFT-A and IFT-B subcomplexes which involved in the various functions of the flagellum,so the study of the function of IFT proteins is essential.Mutations in all six genes encoding IFT-A polypeptides cause skeletal anomalies accompanied with multiple organ defects.To assess the working mechanism of IFT-A complex in IFT and ciliogenesis,we have analyzed an ift43 “null” mutant of Chlamydomonas along with the rest IFT-A mutants identified through RESDA based PCR cloning strategy.IFT43 loss or partial deletion of the IFT43 pfam sequence leads to cells with no or short flagella.IFT43 pfam sequence is necessary and sufficient for IFT43's function.Surprisingly,ift43 mutants flagella accumulate IFT-B as expected,but however also accumulate more IFT-A subunits,which is distinct from other IFT-A mutants.The homeostasis of the IFTA protein levels is regulated by the integrity of the IFT-A complex though protein degradation mediated by the loss of IFT43.Combined biochemical pulldown assay and yeast two-hybrid assay have resolved the organization of the IFT-A complex.By immunofluorescence,we showed that the localization of IFT-A complex at the peribasal body region are dependent on the integrity of the complex.Furthermore,flagellar regeneration and elongation experiment indicate that IFT-A is required for transport of ciliary precursors from the cytoplasmic pool to flagella.By using cytoplasmic dynein inhibitor ciliobrevin D on WT cells,we had verified this hypothesis which revealing a new role of IFT-A in the cell body in addition to cilia.