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Cartwheel Architecture of Trichonympha Basal Body

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Science  03 Aug 2012:
Vol. 337, Issue 6094, pp. 553
DOI: 10.1126/science.1222789

Abstract

Centrioles and basal bodies are essential for the formation of cilia, flagella, and centrosomes. They exhibit a characteristic ninefold symmetry imparted by a cartwheel thought to contain rings of SAS-6 proteins. We used cryoelectron tomography to investigate the architecture of the exceptionally long cartwheel of the flagellate Trichonympha. We found that the cartwheel is a stack of central rings that exhibit a vertical periodicity of 8.5 nanometers and is able to accommodate nine SAS-6 homodimers. The spokes that emanate from two such rings associate into a layer, with a vertical periodicity of 17 nanometers on the cartwheel margin. Thus, by using the power of biodiversity, we unveiled the architecture of the cartwheel at the root of the ninefold symmetry of centrioles and basal bodies.

Centrioles and basal bodies are evolutionarily conserved organelles that are essential for the formation of cilia, flagella, and centrosomes and characterized by a ninefold radial symmetric arrangement of microtubules. A structure called the cartwheel serves as a scaffold for this arrangement and exhibits a central hub ~22 nm in diameter, from which nine spokes emanate toward peripheral microtubules (1). SAS-6 proteins are essential for cartwheel assembly across evolution and harbor a globular N-terminal domain followed by a coiled coil (1). SAS-6 proteins undergo homodimerization and oligomerization between homodimers (2, 3). In vitro, this results in macromolecular assemblies and ringlike structures ~22 nm in diameter (3), but whether such rings exist in vivo is unclear. Moreover, how SAS-6 oligomers give rise to the ~100-nm-high cartwheel observed in most species is not understood. Conceivably, cartwheel height could be achieved via a helical or a stacking mechanism (4). An architectural model is needed to distinguish between these possibilities and unravel mechanisms of cartwheel assembly.

We sought to obtain a three-dimensional (3D) map of the cartwheel in the native state by using cryoelectron tomography (cryo-ET). The ~100-nm height of the cartwheel in most species and its instability (5) precluded such analysis. To circumvent this limitation, we turned to Trichonympha flagellates that live as symbionts in the gut of termites and have exceptionally elongated basal bodies, with ~1500-nm-high cartwheels (6). Apart from its exceptional height, the overall organization of the Trichonympha cartwheel is similar to that in other species (Fig. 1A and fig. S1).

Fig. 1

Cartwheel architecture. (A) Cross-section of chemically fixed Trichonympha cartwheel, with the central hub from which emanate nine spokes. Scale bar, 40 nm. (B) Longitudinal section of Trichonympha basal body cryotomogram. Only about a quarter of the basal body is shown. Scale bar, 40 nm. (C) Higher magnification of the region boxed in (B), illustrating the ~8.5-nm periodicity of the central hub. Scale bar, 10 nm. (D) Low-threshold 3D electron microscopy (EM) map of the central hub, with ~8.5-nm periodicity between central rings. (E) Top and side views of structural model of Bld12p (Chlamydomonas SAS-6) ring (red) fitted in the high-threshold 3D EM map of the central hub. (F) 3D representation of a cartwheel segment. Each layer consists of two central rings ~8.5 nm apart joined by their spokes toward the periphery, thus generating the ~17-nm periodicity at the margin.

We purified Trichonympha basal bodies and analyzed them by using cryo-ET (Fig. 1B and movie S1). This revealed that the central hub exhibits a vertical periodicity of ~8.5 nm (Fig. 1C). Two-dimensional Fourier analysis of tomogram projections yielded two main layer lines at 1/170 and 1/85 Å (fig. S2A). A single layer line was detected at 1/85 Å by retaining only the signal from the central hub (fig. S2B), whereas the cartwheel margin gave rise to the 1/170 Å layer line (fig. S2C).

We then used 3D translational and rotational autocorrelation to address whether the central hub is a helix or a stack (fig. S3A). We found a translation peak every ~8.5 nm (fig. S3B), whereas rotational analysis revealed a ninefold symmetry with correlation peaks every 40° (fig. S3C). Successive rings did not exhibit a measureable twist, indicating that the central hub is not helical but instead a lattice of stacked rings.

We next determined the structure of the central hub by using single-particle image reconstruction (Fig. 1D, fig. S4, and movie S2). Because the N-terminal domains of SAS-6 proteins are structurally conserved (3), we tested whether a ring of the Chlamydomonas SAS-6 protein could fit into the 3D map of the Trichonympha central hub. We found this to be the case, with extra densities between rings suggestive of additional proteins (Fig. 1E, fig. S4A, and movie S3). This supports the view that SAS-6 rings exist in vivo.

We sought to unveil the full architecture of the cartwheel by using subtomogram averaging and ninefold symmetrization, notably to understand how the ~8.5-nm periodicity of the central hub translates into the ~17-nm periodicity at the margin. The resulting 3D structure revealed that radial spokes emanating from the central hub associate two by two ~20 nm thereafter, merging into a single bundle, thus forming layers exhibiting ~17-nm periodicity (Fig. 1F, fig. S5, and movie S4). It will be interesting to investigate to what extent the conclusions we were able to draw by using power of biodiversity apply in other species.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1222789/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S6

References (715)

Movies S1 to S4

References and Notes

  1. Acknowledgments: We thank M. Steinmetz, A.-M. Tassin, and Y. Hongoh for fruitful discussions and comments on the manuscript; J.-D. Rochaix and A. Shapiguzov for Chlamydomonas; and S. Rosset and G. Knott (BioEM core facility, EPFL) for help with EM. Supported by grants from the Swiss National Foundation (Sinergia CRSII3_125463 to P.G.; NF31003A‐125131/1 to T.I.) and the European Research Council (AdG 233335 to P.G.). Additional data and information are in the supplementary materials.
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