A structural road map to unveil basal body composition and assembly

Abstract

EMBO J 31 3, 552–562 (2012); published online December 13 2011

The Basal Body (BB) acts as the template for the axoneme, the microtubule-based structure of cilia and flagella. Although several proteins were recently implicated in both centriole and BB assembly and function, their molecular mechanisms are still poorly characterized. In this issue of The EMBO journal, Li and coworkers describe for the first time the near-native structure of the BB at 33 Å resolution obtained by Cryo-Electron Microscopy analysis of wild-type (WT) isolated Chlamydomonas BBs. They identified several uncharacterized non-tubulin structures and variations along the length of the BB, which likely reflect the binding and function of numerous macromolecular complexes. These complexes are expected to define BB intrinsic properties, such as its characteristic structure and stability. Similarly to the high-resolution structures of ribosome and nuclear pore complexes, this study will undoubtedly contribute towards the future analysis of centriole and BB biogenesis, maintenance and function.

The microtubule (MT)-based structure of the cilium/flagellum grows from the distal part of the Basal Body (BB), which in many animal cells develops from the mature centriole in the centrosome. Electron microscopic (EM) images of chemically fixed resin-embedded centrioles and basal bodies (CBBs) suggest that their ultrastructure is similar, and that their key components are MTs. The mechanisms underlying the organization of CBB MTs, comprising highly stable closed and open MTs, are likely to hold many surprises as they are remarkably different from other microtubular structures in the cell. Additionally, non-MT-based structures are also part of the CBB, including a cartwheel in the proximal lumen region that reinforces CBB symmetry (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011).

Several centriole components and BB proteins were identified by comparative and/or functional genomics and proteomics studies of purified CBBs (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011). Advances in our understanding of the molecular mechanisms of CBB assembly depend on high-resolution comparative studies of wild-type (WT) and mutant structures, as well as characterization of the localization of molecular complexes within the small CBB structure. Despite the existence of beautiful ultrastructure data acquired from chemically fixed specimens (Geimer and Melkonian, 2004; Ibrahim et al, 2009), high-resolution structures of native CBBs were missing. Using electron cryo-tomography and 3D subtomogram averaging, Li et al (2012) solved the structure of the near-native BB triplet at 33 Å resolution. A pseudo-atomic model of the tubulin protofilaments at the core of the triplets was built by fitting the atomic structure of α/β-tubulin monomers into the BB tomograms.

The 3D density map reveals several additional densities that represent non-tubulin proteins attached, both internally and externally, to all triplet MTs, some linking MTs inside the triplets and/or MTs in consecutive triplets (Li et al, 2012; for a summary, see Table I). These structures are likely composed of several proteins that have previously been isolated with CBBs. A Y-shaped structure and large rod-shaped structures emanate from the triplet A/B- and C-tubules, respectively, and extend towards the BB central lumen. Possibly, these large inner circular structures in the BB lumen function as a scaffold that stabilizes the entire BB barrel (Li et al, 2012; Table I). Linker structures had been observed before (Geimer and Melkonian, 2004; Ibrahim et al, 2009), but with less detail and complexity. The authors speculate that some of the additional densities present at the A- and B-tubule inner wall might correspond to proteins of the tektin family, probably conferring rigidity to the BB triplet (Amos, 2008).

Table 1. Characteristics of the non-α/β-tubulin structures reported in Li et al (2012) in this issue of The EMBO journal.

The authors also show that the BB proximal and distal structures are significantly different. The majority of the changes are confined to (1) the C-tubule, (2) linkers between the adjacent triplets and (3) the twist angle of the triplets along the BB length (Li et al, 2012; Table I; Figure 1). It is possible that together with the cartwheel, the linkers between consecutive triplets contribute to establishing and reinforcing the CBB nine-fold symmetry, by defining the angles between triplets and in consequence the available space to fit these MTs. The authors also propose that the structural variations along the length of the BB suggest a sequential and coordinated BB assembly process. It will be important to obtain high-resolution structures of the growing WT CBB and of mutants in genes associated with CBB stability and elongation, such as δ-tubulin, POC5, CPAP, POC1 and Bld10 (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011) to complement previous work (Pelletier et al, 2006; Guichard et al, 2010) and to unveil CBB assembly mechanisms.

Figure 1.

Proximal and distal views of the reconstructed basal body model. MT triplets are represented in blue and non-tubulin proteins attached to the triplets are represented in yellow. Note the structural differences between the proximal and distal regions of the BB at the level of the C-tubule and non-tubulin structures. Lower images represent 3 × magnified view of the box marked area; white arrowheads—indicate the changes in the C-Tubule configuration; black arrowheads—indicate changes in the non-MT structures. Distal view is mirrored to facilitate the comparison with proximal view. Images were kindly provided by Sam Li.

A comparison of the BB structure with that of the axoneme (resolved at 30 Å; Sui and Downing, 2006) revealed that the distribution of the accessory structures on the outer and inner surface of the A- and B-tubules of the BB triplet are different from the axonemal doublet MTs for which they serve as template (Li et al, 2012). It will be important in the future to understand what those differences mean for CBB and axoneme function, including links with pericentriolar components and motility.

The high-resolution structure of ribosome and nuclear pore complexes, solved by single particle reconstruction electron cryo-tomography, contributed immensely to our knowledge on these organelles assembly and function (reviewed in Ramakrishnan, 2009 and Ben-Harush et al, 2010). The BB high-resolution structural analysis reported in this article (Li et al, 2012) will certainly pave the road for the identification of essential non-MT BB components, and allow us to understand their molecular role in the context of CBB biogenesis, maintenance and function.