Three-dimensional structure of basal body triplet revealed by electron cryo-tomography

The Howard Hughes Medical Institute, University of California, San Francisco, CA 94158-2517, USA.
The EMBO Journal (Impact Factor: 10.43). 12/2011; 31(3):552-62. DOI: 10.1038/emboj.2011.460
Source: PubMed


Basal bodies and centrioles play central roles in microtubule (MT)-organizing centres within many eukaryotes. They share a barrel-shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo-tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo-atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non-tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ-tubulin and other components participate in the assembly of the basal body.

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    • "Our structural studies offer an intriguing possibility that integrates the aforementioned properties into a mechanistic role for CPAP in the centriole ultrastructure. A previous cryo-tomographic study of purified C. reinhardtii basal bodies showed distinct electron densities in the vicinity of the microtubule wall (Li et al., 2012). We propose that CPAP accounts for an elongated density observed there that lies roughly parallel to the microtubule triplet (Figures 4A and 4B). "
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    ABSTRACT: Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal "strut" that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections.
    Structure 09/2013; 21(11). DOI:10.1016/j.str.2013.08.019 · 5.62 Impact Factor
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    • "We supposed that if certain residues are important for the lateral interactions of the MT, they should be well conserved in b-tubulin. They also may be conserved in a-and g-tubulins, but not in d-and ε-tubulins, because the former establish lateral interactions similar to those of b-tubulin (Kollman et al., 2010, 2011), but the latter do not (Inclá n and Nogales, 2001; Li et al., 2012). Therefore, two criteria were considered, the conservation (C) of the residues and their selectivity (S), i.e., their conservation only in a, b, and g-tubulins. "
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    ABSTRACT: A microtubule is a dynamic system formed of αβ-tubulins. The presence of nonhydrolyzable guanosine-5'-triphosphate (GTP)/guanosine diphosphate (GDP) on the β-tubulins provokes microtubule polymerization/depolymerization. Despite the large number of experimental studies of this dynamical process, its mechanism is still unclear. To provide insights into this mechanism we studied the first depolymerization steps of GDP/GTP-bound microtubules by normal-mode analysis with the all-atom model. We also constructed a depolymerizing microtubule and compared it to cryo-electron microscopy tomograms (cyro-ET). The results show that during depolymerization, the protofilaments not only curve but twist to weaken their lateral interactions. These interactions are stabilized by GTP, but not evenly. Not all of the interface residues are of equal importance: five of them, belonging to the H2-S3 loop, play a special role; acting as a lock whose key is the γ-phosphate of GTP. Sequence alignments of several tubulins confirm the importance of these residues.
    Structure 04/2013; 21(5). DOI:10.1016/j.str.2013.03.009 · 5.62 Impact Factor
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    • "Moreover, the importance of centrioles/basal bodies and centrosomes for human health and disease is well recognized (Bettencourt-Dias et al., 2011; Doxsey et al., 2005b; Nigg, 2002; Nigg and Raff, 2009). In contrast, although recent cryo-electron tomography has revealed important structural information on centriole architecture (Guichard et al., 2010; Li et al., 2012), definitive information about the spatial organization of the various components remains scarce. Conventional light microscopy has been widely used to determine the approximate localization of centrosomal proteins, but the dimensions of centrioles are close to the Abbe-Rayleigh diffraction limit of optical resolution, that is at best 200 nm in the lateral and 500 nm in the axial direction (Schermelleh et al., 2010). "
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    ABSTRACT: Centrioles are essential for the formation of cilia and flagella. They also form the core of the centrosome, which organizes microtubule arrays important for cell shape, polarity, motility and division. Here, we have used super-resolution 3D-structured illumination microscopy to analyse the spatial relationship of 18 centriole and pericentriolar matrix (PCM) components of human centrosomes at different cell cycle stages. During mitosis, PCM proteins formed extended networks with interspersed γ-Tubulin. During interphase, most proteins were arranged at specific distances from the walls of centrioles, resulting in ring staining, often with discernible density masses. Through use of site-specific antibodies, we found the C-terminus of Cep152 to be closer to centrioles than the N-terminus, illustrating the power of 3D-SIM to study protein disposition. Appendage proteins showed rings with multiple density masses, and the number of these masses was strongly reduced during mitosis. At the proximal end of centrioles, Sas-6 formed a dot at the site of daughter centriole assembly, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but Cep135 was associated mostly with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation. Our study provides novel insights into the architecture of human centrosomes and illustrates the power of super-resolution microscopy in revealing the relative localization of centriole and PCM proteins in unprecedented detail.
    Biology Open 10/2012; 1(10):965-76. DOI:10.1242/bio.20122337 · 2.42 Impact Factor
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Wallace Marshall