FBB18 Regulates Cilia Motility And Comparative Proteomic Analysis Of Disassembling Cilia

Cilia are conserved organelles protruding from cell surface.Cilia consist of axonemes,matrix and membrane.Newly synthesized proteins in cell body are transported into cilia depending on intraflagella transport(IFT).Eukaryotic cilia function in regulation of cell growth,cell differentiation and development involving in cell motility,fluid flow and signal transduction.Defects in cilia assembly,disassembly and motility impair normal ciliary functions,resulting in a series of human diseases named ciliopathies.Thus,researches on mechanism of ciliary motility,ciliary assembly and disassembly not only help understand cilia characteristics per se,but also provide novel clues and theoretical basis for diagnosis and therapy of ciliopathies.FBB18 is conserved in organisms with motile cilia and was reported to be one of axoneme dynein arm preassembly factors in human and zebrafish,however,the mechanism is still unclear.We isolated a null mutant of FBB18 in Chlamydomonas using insertional mutagenesis.FBB18 mainly localized in cytoplasm with only a little flagellar localization.Electron-microscope of fbb18 mutant cilia showed that both inner and outer dynein arms were reduced or missing.Of particular,site for DHC? in the remained ODAs was missing completely.N-terminal coiled-coil domain and C-terminal DUF2870 domain were both essential for function of FBB18.Absence or reduced dynein arm heavy chains in both fbb18 mutant cilia and cell body showed that dynein arm defects in fbb18 was likely caused by disruption of cytoplasmic assembly.Further,we identified a few candidate interactors by immunoprecipitation and tandem mass spectrometry.Yeast-two hybrid and immunoprecipitation verified the conserved direct interaction of FBB18 and another preassembly factor ODA7.We further identified direct interaction of FBB18 with other reported dynein arm assembly factors including PF13,MOT48 and WDR92.Taking together,we reported a conserved protein FBB18 involving in regulation of dynein arm preassembly in cytoplasm and proved that FBB18 may function as an adaptor interacting with other factors to assist folding of dynein arm heavy chains and complex formation.Ciliary disassembly plays important role in cell cycle and ciliary diseases.To get some signaling pathway information and have an overview of ciliary disassembly,we analysed proteome of disassembling cilia of Chlamydomonas.Ninety one proteins were found upregulated more than 1.4 fold.The proteins of the IFT machinery not only increased but also exhibited stoichiometric changes.Other proteins increase in disassembling cilia include signaling molecules,chaperones,proteins involved in microtubule dynamics or stability.Particularly,we have identified a ciliopathy protein C21orf2,the AAA-ATPase CDC48 and stabilizers of axomemal microtubules including FAP203 and FAP236.Proteomic analysis of disassembling cilia provide new insights into understanding of ciliary disassembly,and likely ciliopathy.