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.

Download full-text


Available from: Mathias Uhlen,
  • Source
    • "(Cep85), also known as coiled-coil domain-containing protein 21 (CCDC21), was a particular interesting candidate. It was initially identified with other 21 proteins as new centrosomal components by complementary proteomics strategies (Jakobsen et al., 2011). Except for its spindle pole association, to date, no other studies have been performed. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nek2 has been implicated in centrosome disjunction at the onset of mitosis to promote bipolar spindle formation, and hyperactivation of Nek2 leads to the premature centrosome separation. Its activity, therefore, needs to be strictly regulated. In this study, we report that Cep85, an uncharacterized centrosomal protein, acts as a binding partner of Nek2A. It colocalizes with isoform A of Nek2 (Nek2A) at centrosomes and forms a granule meshwork enveloping the proximal ends of centrioles. Opposite to the effects of Nek2A, overexpression of Cep85 in conjunction with inhibition of the motor protein Eg5 (also known as KIF11) leads to the failure of centrosome disjunction. By contrast, depletion of Cep85 results in the precocious centrosome separation. We also define the Nek2A binding and centrosome localization domains within Cep85. Although the Nek2A-binding domain alone is sufficient to inhibit Nek2A kinase activity in vitro , both domains are indispensable for full suppression of centrosome disjunction in cells. Thus, we propose that Cep85 is a bona fide Nek2A-binding partner that surrounds the proximal ends of centrioles where it cooperates with PP1γ (also known as PPP1CC) to antagonize Nek2A activity in order to maintain the centrosome integrity in interphase in mammalian cells.
    Journal of Cell Science 09/2015; 128(17). DOI:10.1242/jcs.171637 · 5.43 Impact Factor
  • Source
    • "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 "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Experimental spatial proteomics, i.e the high-throughput assignment of proteins to sub-cellular compartments based on quantitative proteomics data, promises to shed new light on many biological processes given adequate computational tools. Here we present pRoloc, a complete infrastructure to support and guide the sound analysis of quantitative mass-spectrometry based spatial proteomics data. It provides functionality for unsupervised and supervised machine learning for data exploration and protein classification and novelty detection to identify new putative sub-cellular clusters. The software builds upon existing infrastructure for data management and data processing. pRoloc is implemented in the R language and available under an open-source license from the Bioconductor project ( A vignette with a complete tutorial describing data import/export and analysis is included in the package. Test data is available in the companion package pRolocdata.
    Bioinformatics 01/2014; 30(9). DOI:10.1093/bioinformatics/btu013 · 4.98 Impact Factor
Show more