Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers

Science

Volumn 283, Issue 5408, 1999, Pages 1733-1735

  Avides, Maria Do Carmo ^a   Glover, David M ^a  

^a UNIVERSITY OF DUNDEE   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

DNA; MICROTUBULE ASSOCIATED PROTEIN; POTASSIUM IODIDE; PROTEIN ASP; TUBULIN; UNCLASSIFIED DRUG;

ANIMAL TISSUE; ARTICLE; CELL NUCLEUS; CENTROSOME; CONTROLLED STUDY; DROSOPHILA MELANOGASTER; EMBRYO; GENE MUTATION; MICROTUBULE; MITOSIS SPINDLE; NONHUMAN; PRIORITY JOURNAL;

ANIMALS; ANTIBODIES; CELL FRACTIONATION; CENTROSOME; DROSOPHILA MELANOGASTER; DROSOPHILA PROTEINS; FLUORESCENT ANTIBODY TECHNIQUE; MICROTUBULE-ASSOCIATED PROTEINS; MICROTUBULES; MITOSIS; MITOTIC SPINDLE APPARATUS; MUTATION; TUBULIN;

EID: 0033548590     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5408.1733     Document Type: Article

References (27)

1. T. Steams and M. Kirschner, Cell 76, 623 (1994).
2. Y. Zheng, M. L. Wong, B. Alberts, T. Mitchison, Nature 378, 578 (1995).
3. M. Moritz, M. B. Braunfeld, J. W. Sedat, B. Alberts, D. A. Agard, ibid., p. 638.
4. S. J. Doxsey, P. Stein, L. Evans, P. D. Calarco, M. Kirschner, Cell 76, 630 (1994).
5. J. B. Dictenberg et al., J. Cell Biol. 141, 163 (1998).
6. R. D. Saunders, M. C. Avides, T. Howard, C. Gonzalez, D. M. Clover, ibid. 137, 881 (1997).
7. P. Ripoll, S. Pimpinelli, M. M. Valdivia, J. Avila, Cell 41, 907 (1985).
8. J. Casal, C. Gonzalez, F. Wandosell, J. Avila, P. Ripoll, Development 108, 251 (1990).
9. Immunostaining of Drosophila brains was carried out as previously described (12). DNA was stained with TOTO3 (Molecular Probes) according to the suppliers instructions. Mitotic spindles were visualized by incubation with anti-α-tubulin monoclonal antibody (done YL 1/2 from Harlan Sera Labs) diluted 1:10. We detected γ-tubulin, using the monoclonal antibody from done GTU88 (Sigma) at a 1:10 dilution. Anti-Asp was polydonal rabbit serum Rb3133 (6) diluted 1:50. Secondary antibodies were purchased from Jackson Immunochemicals and used according to the supplier's instructions. Preparations were visualized in a Bio-Rad 1024 confocal scanning head in conjunction with a Nikon Optiphot microscope.
10. The immunolocalization of Asp on the telophase spindle in cells of the larval brain is not shown. Asp is found in the region between the nuclei and the central spindle.
11. dd8 (23).
12. C. Gonzalez et al., J. Cell Sci. 96, 605 (1990).
13. Centrosomes were purified from Drosophila embryos according to published protocols for the preparation of centrosomes from Chinese hamster ovary cells (24). The final centrifugation was through a 20 to 62.5% (w/w) sucrose gradient over a 70% sucrose cushion in a SW27 (Beckman) rotor for 90 min at 65,000g at 4°C. Microtubule nudeation assays, extraction with 1 M Kl, and complementation assays were carried out as previously described (17). Tubulin and rhodamine-labeled tubulin used in the centrosome nucleation assays were purchased from Molecular Probes.
14. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels were blotted to polyvinylidene difluoride membranes (Millipore). The following antibodies were used: anti-Asp, Rb3133 (6), anti-KLP61F (25); anti-Polo, MA294 (26); anti-γ-tubulin. GTU88 (Sigma); and anti-β-tubulin, BX69 (26). All primary antibodies were diluted 1:500, with the exception of MA294 and BX69, which were diluted 1:4. Peroxidase-conjugated secondary antibodies were purchased from Jackson lmmunochemicals and used according to the suppliers instructions. Bound antibodies were detected by chemiluminescence using chemicals from Amersham (ECL) or Pierce (Supersignal).
15. A number of control antibodies have been used, including one against the centrosomal component CP190. None of these would prevent microtubule nucleation by the centrosome preparation.
16. B. J. Schnackenberg, A. Khodjakov, C. L. Rieder, R. E. Palazzo, Proc. Natl. Acad. Sci. U.S.A. 95, 9295 (1998).
17. M. Moritz, Y. Zheng, B. Alberts, K. Oegema, J. Cell Biol. 142, 775 (1998).
18. Asp was immunodepleted from 100 μ1 of Drosophila embryo extract by incubation with affinity-purified anti-Asp (20 to 50 μg) coupled to protein G-Sepharose beads (Sigma).
19. Microtubules were polymerized from Drosophila embryo extracts by addition of guanosine triphosphate (GTP) and taxol (6). The microtubule pellet was sequentially extracted with 500 mM NaCl, 750 mM NaCl, and 1 M Kl. Soluble fractions were concentrated by ultrafiltration using Millipore Ultrafree systems.
20. M. C. Avides and D. M Glover, data not shown.
21. R. Heald et al., Nature 382, 420 (1996).
22. R. Heald, R. Tournebize, A. Habermann, E. Karsenti, A. Hyman, J. Cell Biol. 138, 615 (1997).
23. H. White-Cooper, M. Carmena, C. Gonzalez, D. M. Glover, Genetics 144, 1097 (1996).
24. T. J. Mitchison and M. W. Kirschner, Methods Enzymol. 134, 261 (1986).
25. N. R. Barton, A. J. Pereira, L. S. Goldstein, Mol. Biol. Cell 6, 1563 (1995).
26. A. Tavares, D. M. Glover, C. E. Sunkel, EMBO J. 15, 4873 (1996).
27. We are grateful to P. Ripoll for stimulating our interest in asp through the original observations of his laboratory and for his continued encouragement. We thank A. Desai, M. Moritz, and B. Lange for their valuable advice about protocols for centrosome purification and microtubule nudeation. We are grateful to L. Goldstein for the antibody to KLP61F. M.C.A. held an European Molecular Biology Organization fellowship during the initial stages of this work, which was supported by grants from the International Association of Cancer Research and Cancer Research Campaign of Great Britain.