Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
The EMBO Journal (Impact Factor: 10.43). 03/2011; 30(8):1520-35. DOI: 10.1038/emboj.2011.63
Source: PubMed


Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate key signalling events. Despite recent advances, the molecular basis for the plethora of processes coordinated by centrosomes is not fully understood. We have combined protein identification and localization, using PCP-SILAC mass spectrometry, BAC transgeneOmics, and antibodies to define the constituents of human centrosomes. From a background of non-specific proteins, we distinguished 126 known and 40 candidate centrosomal proteins, of which 22 were confirmed as novel components. An antibody screen covering 4000 genes revealed an additional 113 candidates. We illustrate the power of our methods by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads to the further functional characterization of centrosomal proteins.

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Available from: Mathias Uhlen, Sep 30, 2015
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    • "In light of the role of coiled - coil domains in the targeting of proteins to the centrosome ( Cohen and Parry 1990 ; Jakobsen et al . 2011 ) , it is likely that the N - terminal coiled - coil domain of Dlg5 is re - sponsible for its basal body localization . The additional interaction of Dlg5 with Kif7 , which also is prominently localized to the basal body , suggests the possibility that the Dlg5 effect on Smo activity may involve formation or alteration of a Smo complex "
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    ABSTRACT: Binding of the Hedgehog (Hh) protein signal to its receptor, Patched, induces accumulation of the seven-pass transmembrane protein Smoothened (Smo) within the primary cilium and of the zinc finger transcription factor Gli2 at the ciliary tip, resulting ultimately in Gli-mediated changes in nuclear gene expression. However, the mechanism by which pathway activation is communicated from Smo to Gli2 is not known. In an effort to elucidate this mechanism, we identified Dlg5 (Discs large, homolog 5) in a biochemical screen for proteins that preferentially interact with activated Smo. We found that disruption of Smo-Dlg5 interactions or depletion of endogenous Dlg5 leads to diminished Hh pathway response without a significant impact on Smo ciliary accumulation. We also found that Dlg5 is localized at the basal body, where it associates with another pathway component, Kif7. We show that Dlg5 is required for Hh-induced enrichment of Kif7 and Gli2 at the tip of the cilium but is dispensable for Gpr161 exit from the cilium and the consequent suppression of Gli3 processing into its repressor form. Our findings suggest a bifurcation of Smo activity in Hh response, with a Dlg5-independent arm for suppression of Gli repressor formation and a second arm involving Smo interaction with Dlg5 for Gli activation. © 2015 Chong et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 02/2015; 29(3):262-76. DOI:10.1101/gad.252676.114 · 10.80 Impact Factor
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    • "In addition, other novel algorithms are proposed, such as PerTurbo (Courty et al., 2011). We have compared and contrasted these algorithms using reliable marker sets and demonstrate in the package documentation that the driving factor for good classification is reflected in the intrinsic quality of the data itself, i.e. efficient cellular content separation, accurate quantitation (Jakobsen et al., 2011), etc. illustrating the minor importance of the classification algorithm with respect to thorough data exploration and quality control. While the exact algorithm might not be the major reason for a good analysis, it is essential to guarantee optimal application of the algorithm. "
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    Bioinformatics 01/2014; 30(9). DOI:10.1093/bioinformatics/btu013 · 4.98 Impact Factor
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    • "The centriole cylinders are built of nine triplet microtubules (MTs) and have approximate diameters of 150‒200 nm and lengths of 400‒450 nm (Vorobjev and Chentsov, 1980; Paintrand et al., 1992; Bornens, 2012). The PCM is composed of more than 100 different proteins (Andersen et al., 2003; Jakobsen et al., 2011), as indicated by proteomics an- alyses. "
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