Centrosomes: Methods for Preparation

Abstract

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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References

Andersen JS, Wilkinson CJ, Mayor T et al. (2003) Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426: 570–574.

Blackburn GR, Barrau MD and Dewey WC (1978) Partial purification of centrosomes from Chinese hamster ovary cells. Experimental Cell Research 113: 183–187.

Blomberg‐Wirschell M and Doxsey SJ (1998) Rapid isolation of centrosomes. Methods in Enzymology 298: 228–238.

Bornens M and Moudjou M (1999) Studying the composition and function of centrosomes in vertebrates. Methods in Cell Biology 61: 13–34.

Bornens M, Paintrand M, Berges J, Marty MC and Karsenti E (1987) Structural and chemical characterization of isolated centrosomes. Cell Motility and the Cytoskeleton 8: 238–249.

Chrétien D, Buendia B, Fuller SD and Karsenti E (1997) Reconstruction of the centrosome cycle from cryoelectron micrographs. Journal of Structural Biology 120: 117–133.

Dobbelaere J, Josue F, Suijkerbuijk S et al. (2008) A genome‐wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biology 6: 1975–1990.

Dutcher SK (1995) Purification of basal bodies and basal body complexes from Chlamydomonas reinhardtii. Methods in Cell Biology 47: 323–334.

Evans L, Mitchison T and Kirschner M (1985) Influence of the centrosome on the structure of nucleated microtubules. Journal of Cell Biology 100: 1185–1191.

Goshima G, Wollman R, Goodwin SS et al. (2007) Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417–421.

Gosti‐Testu F, Marty MC, Berges J, Maunoury R and Bornens M (1986) Identification of centrosomal proteins in a human lymphoblastic cell line. EMBO Journal 5: 2545–2550.

Gräf R (2001) Isolation of centrosomes from Dictyostelium. Methods in Cell Biology 67: 337–357.

Hilger M, Bonaldi T, Gnad F and Mann M (2009) Systems‐wide analysis of a phosphatase knock‐down by quantitative proteomics and phosphoproteomics. Molecular and Cellular Proteomics 8: 1908–1920.

Hormeno S, Ibarra B, Chichon FJ et al. (2009) Single centrosome manipulation reveals its electric charge and associated dynamic structure. Biophysical Journal 97: 1022–1030.

Keller LC and Marshall WF (2008) Isolation and proteomic analysis of Chlamydomonas centrioles. Methods in Molecular Biology 432: 289–300.

Keller LC, Romijn EP, Zamora I, Yates JR 3rd and Marshall WF (2005) Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary‐disease genes. Current Biology 15: 1090–1098.

Kellogg DR, Moritz M and Alberts BM (1994) The centrosome and cellular organization. Annual Review of Biochemistry 63: 639–674.

Komesli S, Tournier F, Paintrand M et al. (1989) Mass isolation of calf thymus centrosomes: identification of a specific configuration. Journal of Cell Biology 109: 2869–2878.

Lange BM and Gull K (1995) A molecular marker for centriole maturation in the mammalian cell cycle. Journal of Cell Biology 130: 919–927.

Lange BM, Bachi A, Wilm M and Gonzalez C (2000) Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates. EMBO Journal 19: 1252–1262.

Lehmann V, Müller H and Lange BM (2006) Immunoisolation of centrosomes from Drosophila melanogaster. Current Protocols in Cell Biology 29: 3.17.1–3.17.13 (chap. 3: unit 3).

Mitchison TJ and Kirschner MW (1984) Microtubule assembly nucleated by isolated centrosomes. Nature 312: 232–237.

Mitchison TJ and Kirschner MW (1986) Isolation of mammalian centrosomes. Methods in Enzymology 134: 261–268.

Moritz M and Alberts BM (1999) Isolation of centrosomes from Drosophila embryos. Methods in Cell Biology 61: 1–12.

Moritz M, Braunfeld MB, Fung JC et al. (1995) Three‐dimensional structural characterization of centrosomes from early Drosophila embryos. Journal of Cell Biology 130: 1149–1159.

Nogales‐Cadenas R, Abascal F, Diez‐Perez J, Carazo JM and Pascual‐Montano A (2009) CentrosomeDB: a human centrosomal proteins database. Nucleic Acids Research 37: D175–D180.

Paintrand M, Moudjou M, Delacroix H and Bornens M (1992) Centrosome organization and centriole architecture: their sensitivity to divalent cations. Journal of Structural Biology 108: 107–128.

Palazzo RE and Vogel JM (1999) Isolation of centrosomes from Spisula solidissima oocytes. Methods in Cell Biology 61: 35–56.

Pereira G and Schiebel E (1997) Centrosome‐microtubule nucleation. Journal of Cell Science 110(part 3): 295–300.

Roobol A, Havercroft JC and Gull K (1982) Microtubule nucleation by the isolated microtubule‐organizing centre of Physarum polycephalum myxamoebae. Journal of Cell Science 55: 365–381.

Rout MP and Kilmartin JV (1990) Components of the yeast spindle and spindle pole body. Journal of Cell Biology 111: 1913–1927.

Schnackenberg BJ and Palazzo RE (1999) Identification and function of the centrosome centromatrix. Biology of the Cell 91: 429–438.

Tassin AM and Bornens M (1999) Centrosome structure and microtubule nucleation in animal cells. Biology of the Cell 91: 343–354.

Thompson‐Coffe C, Coffe G, Schatten H, Mazia D and Schatten G (1996) Cold‐treated centrosome: isolation of centrosomes from mitotic sea urchin eggs, production of an anticentrosomal antibody, and novel ultrastructural imaging. Cell Motility and the Cytoskeleton 33: 197–207.

Tournier F, Karsenti E and Bornens M (1989) Parthenogenesis in Xenopus eggs injected with centrosomes from synchronized human lymphoid cells. Developmental Biology 136: 321–329.

Wigge PA, Jensen ON, Holmes S et al. (1998) Analysis of the Saccharomyces spindle pole by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. Journal of Cell Biology 141: 967–977.

Further Reading

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

Badano JL, Teslovich TM and Katsanis N (2005) The centrosome in human genetic disease. Nature Reviews. Genetics 6: 194–205.

Bettencourt‐Dias M and Glover DM (2007) Centrosome biogenesis and function: centrosomics brings new understanding. Nature Reviews. Molecular Cell Biology 8: 451–463.

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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How to Cite close
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]