Centrosomes: Methods for Preparation


The centrosome of higher eukaryotic cells is the main microtubule‐organising centre. To understand the molecular mechanisms underlying this organelle's biogenesis and important functions in several cellular processes, such as microtubule nucleation, cell division and stress response, it was critical to develop methods for isolating biochemically meaningful quantities of centrosomes. Centrosomes have been isolated from a variety of organisms and based on these preparations, numerous aspects of this intriguing organelle's morphological, functional and biochemical properties have been uncovered. Better isolation procedures along with the development of new technologies, like RNAi (ribonucleic acid interference) and the increasing accuracy of mass spectrometry and electron microscopy techniques, have profoundly improved our knowledge of the centrosome, leading to a better understanding of its implications in various cellular processes and in diseases.

Key concepts

  • Cultured animal cells, animal tissue, Drosophila embryos, green algae, budding yeast, fungi and surf clams are suitable starting materials for centrosome isolation.

  • Centrosome isolation from animal cells requires cell lysis in low ionic strength buffers.

  • Enrichment of centrosomes is achieved by density centrifugation in sucrose step gradients.

  • Immunoaffinity purification has been employed to further enrich centrosomes from Drosophila embryos.

  • Isolated centrosomes are competent to nucleate microtubules.

  • The analysis of isolated centrosomes has provided a wealth of information on their biochemical composition and novel structural and functional features.

  • Many centrosome components have been identified by applying mass spectrometry and antibody approaches.

  • RNAi has been used to study centrosome functions in cellular signalling pathways and the cell cycle and to identify novel components.

  • Electron tomography will be an informative tool to study the supramolecular structures of isolated centrosomes.

Keywords: centrosome; centriole; microtubule nucleation; isolation; affinity purification; mammalian cells; Drosophila

Figure 1.

This image shows the consecutive isolation steps assayed by immunofluorescence microscopy using an anti‐γ‐tubulin antibody to evaluate number and integrity of isolated centrosomes: (a) centrosomes in embryo homogenate; (b) enriched centrosome fraction from first sucrose gradient; (c) enriched centrosome fraction from second sucrose gradient; (d) immunopurified centrosomes. Centrosomes are shown in yellow and beads are shown in red. Reproduced from Lehmann et al. with permission from Wiley.



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Further Reading

Andersen JS and Mann M (2006) Organellar proteomics: turning inventories into insights. EMBO Reports 7: 874–879.

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Doxsey S (2001) Re‐evaluating centrosome function. Nature Reviews. Molecular Cell Biology 2: 688–698.

Gonzalez C (2008) Centrosome function during stem cell division: the devil is in the details. Current Opinion in Cell Biology 20: 694–698.

Lange BM (2002) Integration of the centrosome in cell cycle control, stress response and signal transduction pathways. Current Opinion in Cell Biology 14: 35–43.

Leis A, Rockel B, Andrees L and Baumeister W (2009) Visualizing cells at the nanoscale. Trends in Biochemical Sciences 34: 60–70.

Nigg EA (ed.) (2005) Centrosomes in Development and Disease. Wiley‐VCH: Weinheim.

Palazzo RE and Davis TN (eds) (2001) Centrosomes and spindle pole bodies. Methods in Cell Biology, vol. 67. San Diego: Academic Press.

Pelletier L, O'Toole E, Schwager A, Hyman AA and Müller‐Reichert T (2006) Centriole assembly in Caenorhabditis elegans. Nature 444: 619–623.

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Habermann, Karin, and Lange, Bodo MH(Apr 2010) Centrosomes: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002597.pub2]