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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    • "Later on, mass spectrometry studies enabled the identification of hundreds of centrosomal components in lymphoblastic cell lines (Andersen et al., 2003; Jakobsen, Schroder, Larsen, Lundberg, & Andersen, 2013), Drosophila (Muller et al., 2010), and mammalian sperm (Firat-Karalar, Sante, Elliott, & Stearns, 2014). Finally, 2D and 3D ultrastructural analyzes of isolated centrosomes, using both transmission electron microscopy and cryo-electron microscopy (cryo-EM) (Figure 1(C)), have enabled better characterization of the overall centriolar structure (Paintrand, Moudjou, Delacroix, & Bornens, 1992), the internal distal part of the centriole (Ibrahim, Messaoudi, Chichon, Celati, & Marco, 2009; Li, Fernandez, Marshall, & Agard, 2012), and the cartwheel (Guichard et al., 2012), as well as the first duplication steps of human centrioles (Guichard, Chretien, Marco, & Tassin, 2010). "
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    ABSTRACT: Centrosomes act as the main microtubule-organizing centre of animal cells and play critical roles in the cell, such as mitotic spindle organization, cell polarity, and motility. They are composed of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar matrix. In mammalian cells, the two centrioles differ structurally due to generational difference, the oldest one bearing appendages which allow the transient docking of the centriole at the plasma membrane in order to grow a primary cilium. Centrosome components are highly conserved throughout evolution and several pathologies have been associated with centrosomal defects. The understanding of such a complex organelle has therefore been a challenge for many researchers and has led to the development of centrosomal purification procedures to assess molecular composition, biological function, and structural organization of centrosomes. In this paper, we detail a step-by-step procedure to generate high yield of purified centrosome obtained from various mammalian cell lines. Copyright © 2015 Elsevier Inc. All rights reserved.
    Methods in cell biology 07/2015; 129:171-89. DOI:10.1016/bs.mcb.2015.03.004 · 1.42 Impact Factor
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    • "Morphological analysis of centrioles from several species (Li et al., 2012; Guichard et al., 2013) reveals conserved structural features, and, other than the ninefold symmetry of centrioles, which is dictated by the structure of SASS6 (Gönczy, 2012), these features have no known molecular basis. For example, cryotomography and 3D- reconstruction studies of Chlamydomonas (Li et al., 2012) and Trichonympha centrioles (Guichard et al., 2013) show nontubulin densities associated with the lumen of microtubule doublets and triplets, and with the linker between them. "
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    ABSTRACT: Centrioles are evolutionarily conserved microtubule-based structures at the core of the animal centrosome that are essential for nucleating the axoneme of cilia. We hypothesized that centriole proteins have been under-represented in proteomic studies of the centrosome, because of the larger amount of pericentriolar material making up the centrosome. In this study, we have overcome this problem by determining the centriolar proteome of mammalian sperm cells, which have a pair of centrioles but little pericentriolar material. Mass spectrometry of sperm centrioles identifies known components of centrioles and many previously uncharacterized candidate centriole proteins. Assessment of localization of a subset of these candidates in cultured cells identified CCDC113, CCDC96, C4orf47, CCDC38, C7orf31, CCDC146, CCDC81 and CCDC116 as centrosome-associated proteins. We examined the highly conserved protein CCDC113 further and found that it is a component of centriolar satellites, is in a complex with the satellite proteins HAP1 and PCM1, and functions in primary cilium formation.
    Journal of Cell Science 07/2014; 127(19). DOI:10.1242/jcs.157008 · 5.43 Impact Factor
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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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