Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates

To determine the molecular composition of the centrosome of a higher eukaryote, we carried out a systematic nano-electrospray tandem or MALDI mass spectrometry analysis of the polypeptides present in highly enriched preparations of immunoisolated Drosophila centrosomes. One of the proteins identified is Hsp83, a member of the highly conserved Hsp90 family including chaperones known to maintain the activity of many proteins but suspected to have other essential, unidentified functions. We have found that a fraction of the total Hsp90 pool is localized at the centrosome throughout the cell cycle at different stages of development in Drosophila and vertebrates. This association between Hsp90 and the centrosome can be observed in purified centrosomes and after treatment with microtubule depolymerizing drugs, two criteria normally used to define core centrosomal components. Disruption of Hsp90 function by mutations in the Drosophila gene or treatment of mammalian cells with the Hsp90 inhibitor geldanamycin, results in abnormal centrosome separation and maturation, aberrant spindles and impaired chromosome segregation. This suggests that another role of Hsp90 might be to ensure proper centrosome function

A Flexible Multiwell Format for Immunofluorescence Screening Microscopy of Small-Molecule Inhibitors.

Large-scale screens in mammalian cells demand for flexible high-throughput screening platforms that allow to analyze cellular traits on a genome-wide level or to identify small-molecule inhibitors (SMIs) from complex compound libraries. In this study we developed and tested high-density cell arrays made out of polydimethylsiloxane (PDMS) that support cell growth directly on standard glass microscope objective slides. We analyzed the effect of 3 reference inhibitors (MLN-8054, VX-680, and flavopiridol) and 4 exploratory, cell permeable small-molecule kinase inhibitors (two benzothiophene-based and two 4-amino-6-arylpyrimidine-based compounds) on different cell lines, using prototype 5 × 5 and 9 × 9 array carpets. We found that high-density PDMS cell arrays support growth of a broad range of cell types, are well suited for compound screens, and are compatible with high-content screening platforms. This novel array format is of particular advantage for compound screening to identify SMIs, when a combination of flexibility with respect to culture volume, well density, and high-resolution imaging is required. In addition, we demonstrated the suitability of this format for reverse transfection and siRNA experiments

Gene ontology analysis of the centrosome proteomes of Drosophila and human

The centrosome is a complex cell organelle in higher eukaryotic cells that functions in microtubule organization and is integrated into major cellular signaling pathways.1–3 For example, a tight link exists between cell cycle regulation and centrosome duplication, as centrosome numbers must be precisely controlled to ensure high fidelity of chromosome segregation.4 The analysis of the centrosome's protein composition provides the opportunity for a better understanding of centrosome function and to identify possible links to cellular signaling pathways.5,6 Our proteomics study of the Drosophila centrosome recently identified 251 centrosome candidate proteins that we subsequently characterized by RNAi in Drosophila SL2 cells and classified according to their function in centrosome duplication/segregation, structure maintenance and cell cycle regulation.7 Interestingly, functional characterization of their human orthologous proteins revealed the highest functional conservation in the process of centrosome duplication and separation. To analyze functional and biochemical interdependencies further, we carried out an analysis of the gene ontology (GO) annotation of the identified Drosophila centrosome proteins, as well as of the human centrosome proteome.5 The GO analysis of the group of proteins that did not show a centrosome, chromosome segregation or cell cycle related phenotype in our RNAi assays suggests that these molecules may constitute linker proteins to other cellular signaling pathways. Furthermore, the results of our GO analysis of components of the human and of the Drosophila centrosome reflect the somatic and embryonic origin, respectively, of the isolated centrosomes, implicating the Drosophila centrosome proteins in developmental signaling and cell differentiation