A Forward Genetic Screen and Whole Genome Sequencing Identify Deflagellation Defective Mutants inChlamydomonas, Including Assignment of ADF1 as a TRP Channel

Abstract

With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2. Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone.

Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation inChlamydomonas

“…6D). The adf1 mutation results in a single amino acid change (A1724D) (49). Similarly, the mutation in our study also disrupted the interaction in a similar assay (Fig.…”

“…When calcium is added to detergent-permeabilized cam mutants, however, the cells deflagellate immediately, indicating that CaM is involved in an acid-triggered signaling pathway that induces a rise in cytosolic calcium rather than being involved in direct activation of the deflagellation machinery. pH shock-induced calcium entry is required for deflagellation because an adf1 mutant defective in a TRP channel is defective in calcium entry as well as in acid-induced deflagellation (18,49). We have found that CaM interacts with ADF1.…”

“…pH shock-induced elevation of cytosolic calcium comes from 2 resources: entry of extracellular calcium and calcium released from internal stores. Acid-induced calcium entry is mediated by ADF1, a TRP (18,49). Internal calcium release is controlled by the generation of IP3 through PLC activation (18,47,50).…”

“…CaM is a regulator of a variety of ion channels, including TRPs (19). Because gene mutations in ADF1, which encodes a TRP channel, inhibit extracellular calcium entry and deflagellation induced by pH shock (18,49), we wondered whether CaM regulated ADF1 to modulate calcium entry. Treatment of cells with high extracellular calcium induces deflagellation, likely through calcium entry (28,54).…”

MKS-NPHP module proteins control ciliary shedding at the transition zone

“…In this organism, deflagellation is triggered by an increase in intracellular calcium near the base of the flagella [32,33,34] and involves microtubule-severing proteins such as katanin [35,36,37]. Interestingly, proteins involved in this process-such as Flagellar automy 1p (Fa1p), Flagellar automy 2p (Fa2p) belonging to the Never in mitosis A related kinase (Nek) kinase family, and Flagellar associated protein 16 (FAP16)-have been shown to localize at the TZ [37,38,39].…”