Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few proteins have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered TFPs are recruited to the mature basal body pre- and post-basal body duplication with differential expression of TFPs at the assembling new flagellum compared to the existing fixed-length old flagellum of 4 TFPs. RNAi depletion of 17 TFPs revealed 6 were necessary for ciliogenesis and a further 3 were necessary for normal flagellum length. We identified 9 TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and length regulation.

CEP164C regulates flagellum length in stable flagella.

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to "lock" at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections.

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.

Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly.

Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22-74/81 interactions. Structure-based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP-loading is not strictly required.