Genetics of the deflagellation pathway in Chlamydomonas and cloning of Fa1p, a 171 kDa protein essential for axonemal microtubule severing

Microtubule severing is a newly appreciated mechanism for the regulation of microtubule dynamics, yet it remains to be thoroughly characterized. We have taken a genetic approach using Chlamydomonas reinhardtii to better understand this process. Signal-induced deflagellation in Chlamydomonas involves Ca2+-activated breakage of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. In this study, we isolated 13 new deflagellation mutants that can be divided into two phenotypic classes, the Adf- class and the Fa- class. Cells with the Adf - deflagellation phenotype are defective in acid-stimulated Ca2+ influx, but can be induced to deflagellate by treatment with non-ionic detergent and Ca2+. Genetic analyses show that the five new adf mutations, as well as the previously identified adf1 mutation, are alleles of the ADF1 gene. Mutants in the second phenotypic class, the Fa- mutants, fail to deflagellate in response to any known chemical stimulus and are defective in Ca2+-activated microtubule severing. Genetic analysis of these eight new fa strains demonstrated that they define two complementation groups, and one of these contains the previously identified fa1 mutation. Diploid analysis showed that five alleles map to the FA1 gene, whereas four alleles define a novel gene that we have named FA2.;We have identified the product of one of these genes, Fa1p. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca 2+-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca2+-dependent/calmodulin binding motifs. By Western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca2+-activated, Ca 2+-binding protein binds Fa1p, leading ultimately to the activation of axonemal microtubule severing.