The Human Centriolar Protein CEP135 Contains a Two-Stranded Coiled-Coil Domain Critical for Microtubule Binding

, ,
Elsevier, 2016
Journal paper

Centrioles are microtubule-based structures that play important roles notably in cell division and cilium biogenesis. CEP135/Bld10p family members are evolutionarily conserved microtubule-binding proteins important for centriole formation. Here, we analyzed in detail the microtubule-binding activity of human CEP135 (HsCEP135). X-ray crystallography and small-angle X-ray scattering in combination with molecular modeling revealed that the 158 N-terminal residues of HsCEP135 (HsCEP135-N) form a parallel two-stranded coiled-coil structure. Biochemical, cryo-electron, and fluorescence microscopy analyses revealed that in vitro HsCEP135-N interacts with tubulin, protofilaments, and microtubules and induces the formation of microtubule bundles. We further identified a 13 amino acid segment spanning residues 96-108, which represents a major microtubule-binding site in HsCEP135-N. Within this segment, we identified a cluster of three lysine residues that contribute to the microtubule bundling activity of HsCEP135-N. Our results provide the first structural information on CEP135/Bld10p proteins and offer insights into their microtubule-binding mechanism.

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Elsevier, 2016
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Tuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly

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Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. The importance of this architecture has been difficult to decipher notably because of the lack of precise tools to modulate the underlying assembly reaction. Here, we developed monobodies against Chlamydomonas reinhardtii SAS-6, characterizing three in detail with X-ray crystallography, atomic force microscopy and cryo-electron microscopy. This revealed distinct monobody-target interaction modes, as well as specific consequences on ring assembly and stacking. Of particular interest, monobody MBCRS6-15 induces a conformational change in CrSAS-6, resulting in the formation of a helix instead of a ring. Furthermore, we show that this alteration impairs centriole biogenesis in human cells. Overall, our findings identify monobodies as powerful molecular levers to alter the architecture of multi-protein complexes and tune centriole assembly. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. Here, the authors develop monobodies against Chlamydomonas reinhardtii SAS-6 and use X-ray crystallography, atomic force microscopy and cryo-electron microscopy to reveal insights into ring assembly and stacking.

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Chemical Genetic Screen Identifies Natural Products that Modulate Centriole Number

Zhou Fang, Pierre Gönczy, Michele Graciotti, Kai Johnsson

Centrioles are microtubule-based organelles found in most eukaryotic cells and that are critical for the formation of cilia and flagella, as well as of centrosomes in animal cells. The number of centrioles must be strictly regulated in proliferating cells in order to ensure genome integrity upon cell division. Despite their importance, however, the mechanisms governing centriole assembly and number control remain incompletely understood, owing in part to a paucity of available small-molecule compounds for dissection and alteration of the underlying processes. Here we have developed a chemical genetic approach to identify small-molecule compounds capable of modulating centriole numbers in human cells. High-throughput screening of approximate to 2600 natural compounds identified 14 candidate molecules that either diminish (ten compounds) or augment (four compounds) the number of centrioles per cell. We investigated the mechanisms of action of four of these compounds and discovered that two of them potentially reduce centriole number through effects on NF-B signalling. Moreover, we established that one further compound blocks cell cycle progression and probably indirectly causes an augmentation of centriole number. The last compound analysed induces, in addition to excess centrioles, exceptionally long primary cilia-like structures. Overall, our analysis demonstrates that natural products constitute a rich source of tool compounds useful for unravelling and manipulating the mechanisms governing centriole assembly and number control.

Wiley-Blackwell2016

Microtubule

Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27&n

Centriole

In cell biology a centriole is a cylindrical organelle composed mainly of a protein called tubulin. Centrioles are found in most eukaryotic cells, but are not present in conifers (Pinophyta), floweri

Cell division

Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome