CEP290 is a dynamic transition zone protein required for tethering the membrane to the transition zone microtubules, normal intraflagellar transport (IFT), and flagellar assembly
Branch Craige1, Che-Chia Tsao2, Dennis R. Diener2, Yuqing Hou1, Karl Lechtreck1, Joel L. Rosenbaum2, and George B. Witman1
1) Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655 USA
2) Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520 USA
Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. The Chlamydomonas CEP290 homologue, encoded by POC3, is highly conserved (BLAST E = 5e-27). A PCR screen of a library of Chlamydomonas insertional mutants identified a strain in which all but the first 2-4 exons of POC3 are deleted. The mutant cells have defects in flagellar assembly and motility. The mutant phenotype co-segregated with the deletion, and transformation of the mutant with genomic DNA encoding wild-type or HA-tagged CEP290 rescued the mutant phenotype. Immunofluorescence (IF) and immuno-EM localization indicated that CEP290 is located in the transition zone between the outer doublets and the membrane. Ultrastructural analysis revealed defects in the connections between the outer doublets of the transition zone and the flagellar membrane in mutant cells, and the detergent-resistant flagellar membrane that remains attached to wild-type transition zones after detergent extraction was lost in detergent-extracted mutant cells. Some flagella display bulges filled with electron dense material; IF indicated that the bulges contain IFT proteins. Western blots of wild-type and mutant flagella revealed that the mutant flagella contain increased levels of IFT complex B proteins and BBS4, and decreased levels of the IFT complex A subunit IFT139 and polycystin-2. Dikaryon rescue experiments revealed that CEP290 is dynamic and can incorporate into pre-assembled wild-type or CEP290-deficient transition zones. The results indicate that CEP290 is required to tether the flagellar membrane to the transition zone microtubules, and functions in establishing the normal ratio of IFT complex A to B in flagella.
e-mail address of presenting author: branch.craige@umassmed.edu