A key regulatory protein for flagellum length control in stable flagella

Summary Cilia and flagella are highly conserved microtubule-based organelles that have important roles in cell motility and sensing [1]. They can be highly dynamic and short lived such as primary cilia or Chlamydomonas [2] or very stable and long lived such as those in spermatozoa [3] photoreceptors [4] or the flagella of many protist cells [3,4]. Although there is a wide variation in length between cell types, there is generally a defined length for a given cell type [1]. Many unicellular flagellated and ciliated organisms have an additional challenge as they must maintain flagella/cilia at a defined length whilst also growing new flagella/cilia in the same cell. It is not currently understood how this is achieved. A grow-and-lock model was proposed for the maintenance of stable flagella where a molecular lock is applied to prevent flagellum length change after assembly [5]. The molecular mechanisms of how this lock operates are unknown, but could be important in cells where an existing flagellum must be maintained whilst a new flagellum assembles. Here we show that Cep164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei . It is only localised on the transition fibres of basal bodies of fully assembled flagella and missing from assembling flagella. In fact, basal bodies only acquire Cep164C in the third cell cycle after they assemble in trypanosomes. Depletion leads to dysregulation of flagellum growth with both longer and shorter flagella; consistent with defects in a flagellum locking mechanism. By controlling delivery of components into the old assembled flagellum, maintenance of stable flagella can occur but limits further growth. This offers an important explanation for how many eukaryotic unicellular cells maintain their existing flagella whilst growing new ones before these cells divide. This work also reveals additional regulatory roles for Cep164 in eukaryotic organisms.