Isolation of centrosomes from Spisula solidissima oocytes.
We have described methods for the preparation of lysates and isolation of centrosomes from parthenogenetically activated oocytes of the surf clam, S. solidissima. Although oocyte availability is seasonal, between June and August as much as 2 liters of lysate can be generated by a single person. Since lysate can be stored frozen at -80 degrees C with no apparent loss in centrosome-dependent microtubule nucleation, this is a convenient system for year-round experimentation. On average, per milliliter of frozen-stored lysate, 2 or 3 x 10(6) centrosomes can be obtained at 3000- or 4000-fold purification by sucrose-density gradient centrifugation. Centrosome fractions typically contain 6.0 x 10(-12) g of protein per centrosome (Vogel et al., 1997) and 140-200 micrograms of protein is usually obtained from a single run involving six sucrose-density gradients (12 ml of lysate). One person can easily run three preparations in a day, and thus 420-600 micrograms of centrosome protein could be prepared daily. Therefore, based on the effort of one individual, as much as 20-40 mg of centrosome protein could be prepared per year. Another convenient feature of the system is that once centrosomes are isolated, they can be stored in high sucrose media at -80 degrees C for years with little or no loss in microtubule nucleation potential. Once isolated, centrosomes can be used for protein analysis, ultrastructural studies, or in functional reconstitution assays (Vogel, 1997). In addition, these preparations offer the isolation of sufficient quantities of centrosome proteins to be used as antigens for generating centrosome-specific antibodies or for obtaining protein sequence for the purpose of antibody production or the design of oligonucleotide primers for isolating cDNA fragments coding for centrosome proteins. Thus, the preparations described offer a biochemical approach for defining centrosome composition. The methods described for immunofluorescence analysis of asters assembled in lysates offer rapid and convenient preparations for screening antibodies for centrosome localization and specificity. Finally, the ability to prepare large quantities of homogeneous centrosomes should enhance ultrastructural studies since many centrosomes can be sectioned and analyzed simultaneously by EM, avoiding the problem of having to hunt through sections of single cells to find a single centrosome for analysis. In addition, colloidal gold localization studies, using antibodies and EM to pinpoint the relative location of individual proteins, could be carried out on populations of centrosomes in the same preparation simultaneously, thus drastically expanding the quantity of data gathered. In conclusion, the clam oocyte system described here offers the potential for a combined structural and biochemical approach for identification of novel centrosome proteins and elucidation of the molecular basis of microtubule nucleation, centrosome assembly, and centrosome function.
Palazzo
R E
RE
Department of Biochemistry, Cell, and Molecular Biology, University of Kansas, Lawrence 66045, USA.
Vogel
J M
JM
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D018385:Centrosome / Q000276:immunology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification; D008974:Mollusca; D009865:Oocytes
D008869:Microtubule-Associated Proteins