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

Li, Sam; Fernández, Julio F.; Marshall, Wallace F.; Agard, David A.

doi:10.1038/emboj.2011.460

Cited by 135 publications

(203 citation statements)

References 70 publications

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“…A similar value (60–150 nm) was reported in a recent study using 3D‐STORM imaging of stable Sas‐6‐positive structures, likely reflecting cartwheels, in detergent‐extracted human U2OS cells (Keller et al , 2014). Assuming that stacked hubs display a vertical periodicity of 8.5 nm (Kitagawa et al , 2011; Guichard et al , 2012, 2013; Li et al , 2012), a cartwheel thickness of 60–150 nm would thus allow for ~7–17 stacks. These numbers, albeit tentative, are encouragingly close to our estimate of 15–16 stacked hubs per cartwheel.…”

Section: Discussionmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

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Section: Discussionmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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“…By taking advantage of repetitive structures, it is possible to use such periodicities to extract multiple copies of the same structural unit to perform subtomogram averaging. However, this method requires a large number of copies to obtain a robust 3D reconstruction (2000e5000 copies, (Guichard et al, 2012(Guichard et al, , 2013Li et al, 2011)). This represents a prohibitive limitation for small or nonperiodical structures.…”

Section: Discussionmentioning

confidence: 99%

“…• Chlamydomonas basal body microtubule triplet: EMD-5252 (Li et al, 2011) • Trichonympha basal body microtubule triplet: EMD-2330 (Guichard et al, 2013) • Trichonympha cartwheel: EMD-2329 (Guichard et al, 2013) • After alignment, the new averaged map can be visualized using UCSF Chimera (Pettersen et al, 2004).…”

Section: Methods 205mentioning

confidence: 99%

“…This curve is used to calculate new angles, limiting the information during tomogram reconstruction and resulting in low information in the z-axis. The missing wedge effect is compensated in the case of the centriole by taking advantage of the ninefold symmetry of the structure during subtomogram averaging (Bui & Ishikawa, 2013;Guichard et al, 2013;Li et al, 2011). To perform subtomogram averaging, it is crucial to use tomograms of centrioles that have not been compressed.…”

Section: Subtomogram Averaging (Figure 4)mentioning

confidence: 99%

“…With these advances, one can now thoroughly analyze the native structure of centrioles by cryomicroscopy using cryotomography and image processing (Guichard, Chrétien, Marco, & Tassin, 2010;Guichard et al, 2012Guichard et al, , 2013Li, Fernandez, Marshall, & Agard, 2011).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Isolation, cryotomography, and three-dimensional reconstruction of centrioles

Guichard¹,

Hamel²,

Neves³

et al. 2015

Methods in Cell Biology

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Tubulin and Microtubules

Amos

2015

Encyclopedia of Life Sciences

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Microtubules are found in almost all types of eukaryotic cell. Their principal constituents are the highly conserved proteins of the tubulin family, in the form of longitudinal protofilaments of tubulin heterodimers. The conserved lateral interactions produce a characteristic 2D lattice. Tubulin structure and conformational changes are now known to atomic resolution. Its GTP‐dependent dynamic self‐assembly and disassembly is modulated by a host of other proteins. This dynamic activity and a more passive role as tracks for interaction with motor proteins, kinesin and dynein, allow microtubules to perform vital roles in various forms of cell motility. They are an essential component of cell division, provide oriented tracks for the transport of cellular organelles and vesicles and are responsible for the relative positioning of cellular compartments. Thus, they are important targets in the control of cancer cells, as their essential activity is severely affected by a variety of drugs. Key Concepts Tubulin heterodimers, consisting of highly homologous α‐ and β‐tubulin monomers, self‐assemble into a unique lattice with polar longitudinal ‘protofilaments’ The assembled sheet has a slightly variable curvature, producing microtubules, with around 13 protofilaments but not strictly defined unless they are grown from a γ‐tubulin template or given a curvature appropriate to exactly 13 protofilaments by accessory proteins The structure of microtubules has been solved to near‐atomic level both with GTP homologues bound to β‐tubulin and with GDP bound; also a depolymerised protofilament structure in a longitudinally curved conformation is known Hydrolysis of bound GTP enables microtubules to assemble with ‘dynamic instability’, behaviour that depends on stochastic disassembly when the GTP ‘cap’ is lost from a growing end Microtubule behaviour is modified by many different accessory proteins that may act just at the tips or everywhere on the lattice wall To play different roles according to their positions in cells, the properties of microtubules may vary owing to different tubulin isotypes and be modified by a wide range of associated proteins Changes can also vary with time (‘ageing’) as a result of post‐translational modification of tubulin and of some accessory proteins Microtubules are employed in axonemes of cilia and flagella, where the motor protein dynein interacts with them to produce beating while other linking components help control the wave‐form Cytoplasmic microtubules provide tracks for vesicles and other cargoes driven by kinesins and cytoplasmic dynein Mitotic and meiotic spindles are assembled from microtubules, which interact with kinetochores and with multiple motor proteins Microtubules are susceptible to many small molecules that depolymerise or hyperstabilise them and thus serve as anti‐cancer drugs.

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

Cited by 135 publications

References 70 publications

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Isolation, cryotomography, and three-dimensional reconstruction of centrioles

Tubulin and Microtubules

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