Elucidation of the signalling pathway of deflagellation

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The mechanism of deflagellation in the protist Chlamydomonas is the heart of my NSERC-funded research program. Surprisingly, these studies lead to discoveries of general relevance to eukaryotic cell cycle progression. Now, I propose to refocus on deflagellation, where there is every indication that we will make new discoveries of general import. Deflagellation is the shedding of flagella into the environment and is induced by cellular stress. The microtubule core of the flagellum is severed at its base, accompanied by resealing of the plasma membrane over the severed microtubules, thereby retaining the integrity of the cytoplasm. A deflagellated cell can regrow new flagella in ~90 minutes. One stress that causes deflagellation is pH shock, which we have shown works via intracellular acidification and activation of calcium influx at the base of the flagellum. Our genetic screen uncovered the adf1 mutants, which are defective in pH shock-induced deflagellation, but are wild-type for the calcium-activated machinery of microtubule severing. The ADF1 gene has not yet been cloned, but is predicted to encode either the acid-sensor or the calcium-permeant channel. Objective #1 is to identify ADF1 and elucidate its mode of action. We have shown that all of the components that are downstream of calcium in the deflagellation pathway are integral to the microtubule-based core of the flagellum. This result indicates that the severing module is a pre-existing protein complex that senses intracellular calcium and executes microtubule severing. Our genetic screen identified FA1 and FA2 as components of this complex. Objective #2 is to identify the remaining components and to elucidate the mechanism of action of this protein complex.