A biochemical and genetic approach to understand the function of UNI2, a gene encoding a novel basal body protein in Chlamydomonas reinhardtii

Abstract

The unicellular green alga Chlamydomonas reinhardtii is typically biflagellate, but forward genetic screens have identified uniflagellar mutants. All uniflagellar mutants ( uni1, uni2, and uni3 ) contain ultrastructural defects in the basal body or transition zone and preferentially assemble a flagellum from the older basal body. The UNI2 gene encodes a novel coiled-coil protein with a potential homolog in the human genome. We rescued the uni2 mutant phenotype with an HA-epitope tagged gene construct. Immunoblot analysis demonstrated that the Uni2 protein migrates as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phophorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Immunofluorescence staining of the Uni2 protein in interphase cells demonstrated that it localizes to four distinct spots coinciding with the location of basal bodies and probasal bodies Immunogold labeling confirmed Uni2 protein localization on probasal bodies and the distal end of basal bodies at the precise point of triplet to doublet microtubule transition between the basal body and flagellum. Using the Uni2 protein as a marker of basal bodies during the cell cycle, we observed the sequential assembly of new probasal bodies beginning at prophase. Double mutant strains with uni1,uni2 or uni2,uni3 genotypes showed enhanced defects in flagellar assembly. Immunoblot analysis showed that phosphorylation of the Uni2 protein is significantly reduced in uni1 mutant cells but is similar to wild-type levels in uni3 mutant cells. Ultrastructural analysis demonstrated enhanced transition zone defects in the uni1,uni2 double mutant cells. Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition from triplet to doublet microtubules between the basal body and flagellum. The transition defect was correlated with an inability to form axonemes. These mutants provide the first mechanistic insights into the pathway mediating the transition of triplet to doublet microtubules during flagellar assembly and suggest an overlap in the pathways mediating microtubule transition and basal body maturation.