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

Abstract

gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.

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

“…It is known that flagellar paralysis can result from loss of structures such as the PFR (Bastin et al, 1998), axonemal radial spokes and central pair (McKean et al, 2003;Smith and Lefebvre, 1997;Warr et al, 1966;Witman et al, 1978), however all these structures are present in TbPACRG AB RNAi-induced cells. The central pair remains present until more than four outer doublets have been lost, suggesting that this is a secondary effect of loss of multiple outer doublets.…”

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

“…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.…”

Conserved and specific functions of axoneme components in trypanosome motility

“…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).…”