Isolation of nucleation-competent centrosomes from Dictyostelium discoideum.
The centrosome of Dictyostelium discoideum is a box-shaped, layered core structure surrounded by a corona which is made up of dense nodules embedded in amorphous material. It is also known as nucleus-associated body. Because of its tight association with the nucleus the centrosome has resisted so far all attempts for isolation in sufficient purity and quantity for biochemical analysis. Here we report on the large-scale isolation of D. discoideum centrosomes after treatment of nucleus-centrosome complexes with a buffer containing sodium pyrophosphate. Following heparin treatment and a filtration step, centrosomes were further purified by density gradient centrifugation. Immunofluorescence analysis of the isolated centrosomes revealed the presence of the D. discoideum 350-kDa antigen, a centrosomal marker protein, gamma-tubulin, and the D. discoideum homologues of pericentrin, Spc110p, and Cdc31p. The structural integrity of the isolated centrosomes was demonstrated by confocal laser microscopy and electron microscopy. Microtubule nucleation assays with purified pig brain tubulin showed that the isolation procedure did not only preserve the structure but also the functionality of the isolated centrosomes. D. discoideum centrosomes should now become an attractive new model system in addition to, and for comparison with, centriolar centrosomes and yeast spindle pole bodies.
Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität München, Germany. Ralph.Graef@lrz.uni-muenchen.de
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D000818:Animals; D018385:Centrosome / Q000276:immunology / Q000648:ultrastructure; D004023:Dictyostelium; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008870:Microtubules