γ-Tubulin Functions in the Nucleation of a Discrete Subset of Microtubules in the Eukaryotic Flagellum

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Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Comparative genomics of flagellated and non-flagellated eukaryotes provides one way to identify new putative flagellar proteins. We identified the Parkin co-regulated gene, or PACRG, from such a screen. Male mice deficient in PACRG are sterile, but its function has been little explored. The flagellated protozoan parasite Trypanosoma brucei possesses two homologues of PACRG. We performed RNA interference knockdown experiments of the two genes independently and both together. Simultaneous ablation of both proteins produced slow growth and paralysis of the flagellum with consequent effects on organelle segregation. Moreover, using transmission electron microscopy, structural defects were seen in the axoneme, with microtubule doublets missing from the canonical 9+2 formation. The occurrence of missing doublets increased toward the distal end of the flagellum and sequential loss of doublets was observed along individual axonemes. GFP fusion proteins of both PACRG homologues localised along the full length of the axoneme. Our results provide the first evidence for PACRG function within the axoneme, where we suggest that PACRG acts to maintain functional stability of the axonemal outer doublets of both motile and sensory cilia and flagella.

Section: Discussionmentioning

confidence: 99%

The Parkin co-regulated gene product, PACRG, is an evolutionarily conserved axonemal protein that functions in outer-doublet microtubule morphogenesis

Dawe

Farr

Portman

et al. 2005

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“…In addition to the presence of the above proteins, the level of polyglutamylation on α-and β-tubulin is increased on older centrioles (Bobinnec et al, 1998) and on older basal bodies (Lechtreck and Geimer, 2000). Unlike centrioles, daughter basal bodies must mature early in their first cell cycle in order to assemble flagella or cilia and so for example, all basal bodies in the ciliated epithelium of the trachea have the ODF2 epitope (McKean et al, 2003). Therefore, there is a need for an additional maturation event in basal bodies which we refer to as early maturation and which must occur at M/G1 phase of the cell cycle rather than in G2/M phase, as for centrioles.…”

Section: Discussionmentioning

confidence: 99%

Mutations in α-tubulin promote basal body maturation and flagellar assembly in the absence of δ-tubulin

Fromherz

Giddings

Gomez‐Ospina

et al. 2004

We have isolated suppressors of the deletion allele of δ-tubulin, uni3-1, in the biflagellate green alga Chlamydomonas reinhardtii. The deletion of δ-tubulin produces cells that assemble zero, one or two flagella and have basal bodies composed primarily of doublet rather than triplet microtubules. Flagellar number is completely restored in the suppressed strains. Most of the uni3-1 suppressors map to the TUA2 locus, which encodes α2-tubulin. Twelve independent tua2 mutations were sequenced. Amino acids D205 or A208, which are nearly invariant residues in α-tubulin, were altered. The tua2 mutations on their own have a second phenotype - they make the cells colchicine supersensitive. Colchicine supersensitivity itself is not needed for suppression and colchicine cannot phenocopy the suppression. The suppressors partially restore the assembly of triplet microtubules. These results suggest that the δ-tubulin plays two roles: it is needed for extension or stability of the triplet microtubule and also for early maturation of basal bodies. We suggest that the mutant α-tubulin promotes the early maturation of the basal body in the absence of δ-tubulin, perhaps through interactions with other partners, and this allows assembly of the flagella.

“…Hence, proteins present in flagella that were assembled before the onset of RNAi remain present and only disappear according to their rate of turnover. Typically, in the case of silencing of flagellar genes, a protein can be missing in the new flagellum but still be present in the old flagellum (Bastin et al, 2000;Kohl et al, 2003;McKean et al, 2003).…”

Section: Identification and Rnai Silencing Of Trypanosome Axoneme Genesmentioning

confidence: 99%

Conserved and specific functions of axoneme components in trypanosome motility

Branche

Kohl

Toutirais

et al. 2006

151

261

The Trypanosoma brucei flagellum is unusual as it is attached along the cell body and contains, in addition to an apparently conventional axoneme, a structure called the paraflagellar rod, which is essential for cell motility. Here, we investigated flagellum behaviour in normal and mutant trypanosome cell lines where expression of genes encoding various axoneme proteins (PF16, PF20, DNAI1, LC2) had been silenced by RNAi. First, we show that the propulsive wave (normally used for forward motility) is abolished in the absence of outer dynein arms, whereas the reverse wave (normally used for changing direction) still occurs. Second, in contrast to Chlamydomonas - but like metazoa, the central pair adopts a fixed orientation during flagellum beating. This orientation becomes highly variable in central-pair- and outer-dynein-arm-mutants. Third, the paraflagellar rod contributes to motility by facilitating three-dimensional wave propagation and controlling cell shape. Fourth, motility is required to complete the last stage of cell division in both insect and bloodstream stages of the parasite. Finally, our study also reveals the conservation of molecular components of the trypanosome flagellum. Coupled to the ease of reverse genetics, it raises the interest of trypanosomes as model organisms to study cilia and flagella.