Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 +/- 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10(3) electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca(2+), over the thermally induced dynamic structure of the centrosome.

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkII␤. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G 2 /M transition in Drosophila SL2 cells.

This review provides a brief overview of the recent work on centrosome proteomics, protein complex identification and functional characterization with an emphasis on the literature of the last three years. Proteomics, genetic screens and comparative genomics studies in different model organisms have almost exhaustively identified the molecular components of the centrosome. However, much knowledge is still missing on the protein-protein interactions, protein modifications and molecular changes the centrosome undergoes throughout the cell cycle and development. The dynamic nature of this large multi-protein complex is reflected in the variety of annotated subcellular locations and biological processes of its proposed components. Some centrosomal proteins and complexes have been studied intensively in different organisms and provided detailed insight into centrosome functions. For example, the molecular, structural and functional characterization of the γ-Tubulin ring complex (γ-TuRC) and the the discovery of the Augmin/HAUS complex has advanced our understanding of microtubule (MT) capture, nucleation and organization. Surprising findings revealed new functions and localizations of proteins that were previously regarded as bona fide centriolar or centrosome components, e.g. at the kinetochore or in the nuclear pore complex regulating MT plus end capture or mRNA processing. Many centrosome components undergo posttranslational modifications such as phosphorylation, SUMOylation and ubiquitylation that are critical in modulating centrosome function and biology. A wealth of information has recently become available driven by new developments in technologies such as mass spectrometry, light and electron microscopy providing more detailed molecular and structural definition of the centrosome and particular roles of proteins throughout the cell cycle and development.

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