Immunoisolation of Centrosomes from Drosophila melanogaster

Verena Lehmann1, Hannah Müller1, Bodo M.H. Lange1

1 Max Planck Institute for Molecular Genetics, Berlin
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.17
DOI:  10.1002/0471143030.cb0317s29
Online Posting Date:  January, 2006
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Abstract

Classical protocols for the isolation of centrosomes from higher eukaryotic cells are based on enrichment of cell organelles by density gradient centrifugation. Various successful protocols have been described that isolate centrosomes from mammalian tissue culture cells, tissue, clam oocytes, Drosophila, and yeast, to mention only some of the more frequently used sources. The material produced is subsequently used in various assays. These include functional tests such as the microtubule nucleation assay, electron microscopic study of centrosome morphology, and antigen localization; the organelles may also be used for the generation of antibodies. Furthermore, centrosomal preparations have been used for the characterization of their protein composition. The method described here focuses on the isolation of centrosomes from the syncytial stages of the early Drosophila embryo. This is a particularly attractive system because these organelles are not bounded by cellular membranes. Moreover, the abundance of pericentriolar material of these centrosomes produces excellent total protein yields.

Keywords: density gradient centrifugation; Immunoisolation; centrosome; Drosophila; protein complex

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Centrosomes from the Early Syncytial Stages of the Drosophila Embryo Using Sucrose Step Gradient Centrifugation
  • Support Protocol 1: Immunofluorescence Microscopy of Isolated Centrosomes
  • Basic Protocol 2: Immunopurification of Centrosomes with Magnetic Beads
  • Support Protocol 2: Immunofluorescence Microscopy of Immunopurified Centrosomes on Magnetic Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Centrosomes from the Early Syncytial Stages of the Drosophila Embryo Using Sucrose Step Gradient Centrifugation

  Materials
  • Large population of Drosophila (e.g., ∼250,000 strain W118 flies) maintained in large environmental incubator or temperature‐ and humidity‐controlled room with fixed day (12 hr) and night (12 hr) cycles (Greenspan, ; Bonte and Becker, ; Sullivan et al., )
  • Apple juice/molasses agar plates (see recipe)
  • Yeast paste: ordinary baker's yeast dissolved in warm water to form a paste
  • Embryo wash: 0.7% (w/v) NaCl/0.04% (v/v) Triton X‐100
  • 3% (v/v) sodium hypochlorite in embryo wash (see above)
  • Homogenization buffer (HB, see recipe)
  • Liquid N 2
  • 5× BRB80 with 100 mM KCl (see recipe), diluted to 1×
  • 25% (v/v) Triton X‐100
  • 100× protease inhibitor mix (PIM, see recipe)
  • 55% and 70% (w/v) sucrose solutions (see reciperecipes)
  • Fine strainer
  • Filter unit fitted onto vacuum flask
  • Motor‐driven Wheaton homogenizer with tight‐fitting Teflon pestle (60‐ml volume)
  • 50‐ml conical polypropylene centrifuge tubes
  • Refrigerated low‐speed centrifuge
  • Miracloth (Calbiochem)
  • 15‐ml snap‐cap polypropylene tubes for freezing and storage of supernatants
  • 39‐ml thin‐walled polyallomer ultracentrifuge tubes (e.g., Beckman)
  • Large‐volume ultracentrifuge (e.g., Beckman) with swinging‐bucket rotor (e.g., Beckman SW 32)
  • Additional reagents and equipment for assessing purify of centrosome fractions from gradient (see protocol 2)

Support Protocol 1: Immunofluorescence Microscopy of Isolated Centrosomes

  Materials
  • 30‐µl aliquots from Pool 1 and Pool 2 (see protocol 1)
  • PBS‐T: phosphate‐buffered saline (PBS; appendix 2A), pH 7.4, containing 0.003% (v/v) Triton X‐100
  • Methanol, −20°C
  • Primary antibody: anti‐γ‐tubulin (Sigma)
  • Secondary antibody: fluorochrome‐conjugated antibody specific for IgG of the species from which the primary anti‐γ‐tubulin antibody was obtained
  • Mounting medium (e.g., Mowiol from Calbiochem, or 50% v/v glycerol)
  • 20 mg/ml (10×) p‐phenylendiamine in H 2O
  • Nail polish
  • Plastic inserts to support a 11‐mm round glass coverslip (Evans et al., )
  • 15‐ml Corex tubes
  • 11‐mm round glass coverslips (grade 1)
  • Refrigerated centrifuge with swinging‐bucket rotor (e.g., Sorvall HB‐6 or Beckman JS 13.1, or equivalent) and adapters
  • 24‐well tissue culture plates
  • Spatula bent at the tip for removing the plastic insert
  • Needle with bent tip
  • Forceps
  • Microscope slides
  • Epifluorescence microscope equipped with immunofluorescence filters and appropriate optics
NOTE: To avoid staining artifacts, never allow the coverslips to dry out at any point during the following protocol.

Basic Protocol 2: Immunopurification of Centrosomes with Magnetic Beads

  Materials
  • Protein G magnetic bead suspension (Dynal)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.2 M triethanolamine, pH 8.2, with and without 20 mM dimethyl pimelidate dihydrochloride (DMP); prepare fresh
  • Anti‐γ‐tubulin antibody (anti‐peptide antibody; not commercially available; affinity‐purified; Tavosanis et al., )
  • Tris⋅Cl, pH 7.5 ( appendix 2A)
  • PBS ( appendix 2A) containing 0.1% (v/v) Tween 20
  • PBS ( appendix 2A) containing 0.1% (v/v) Tween 20 and 0.02% (w/v) thimerosal
  • Pool 2 of centrosomes (see protocol 1)
  • Dilution buffer (see recipe), 4°C
  • PBS‐T: phosphate‐buffered saline (PBS; appendix 2A), pH 7.4, containing 0.003% (v/v) Triton X‐100, 4°C
  • Methanol, −20°C
  • SDS sample buffer (unit 6.1)
  • Magnetic particle collector (magnet and magnetic stand) suitable for 1.5‐ml microcentrifuge tubes (e.g., Dynal)
  • End‐over‐end rotator
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and determining purity of centrosome preparations from immunomagnetic purification (see Support Protocols protocol 21 and protocol 42)

Support Protocol 2: Immunofluorescence Microscopy of Immunopurified Centrosomes on Magnetic Beads

  Materials
  • Centrosomes immunopurified on magnetic beads (see protocol 3)
  • Methanol, −20°C
  • PBS‐T: phosphate‐buffered saline (PBS; appendix 2A), pH 7.4, containing 0.003% (v/v) Triton X‐100
  • Primary antibody: anti‐γ‐tubulin (Sigma)
  • PBS‐T containing 0.1% (w/v) bovine serum albumin (BSA)
  • Secondary antibody: fluorochrome‐conjugated antibody specific for IgG of the species from which the primary anti‐γ‐tubulin antibody was obtained
  • Mounting medium: Mowiol (Calbiochem)
  • 20 mg/ml (10×) p‐phenylendiamine in H 2O
  • Clear nail polish
  • Magnetic particle collector (magnet and magnet stand) suitable for 1.5‐ml microcentrifuge tubes (e.g., Dynal)
  • End‐over‐end rotator
  • Microscope slides
  • Round coverslips (grade 1, 11 mm diameter)
  • Epifluorescence microscope equipped with immunofluorescence filters and appropriate optics
NOTE: Handle samples with care and avoid vigorous mixing or vortexing.
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Figures

Videos

Literature Cited

Literature Cited
   Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A., and Mann, M. 2003. Characterization of the human centrosome by protein correlation profiling. Nature 426:570‐574.
   Blomberg‐Wirschell, M. and Doxsey, S.J. 1998. Rapid isolation of centrosomes. Methods Enzymol. 298:228‐238.
   Bonte, E. and Becker, P.B. 1999. Preparation of chromatin assembly extracts from preblastoderm Drosophila embryos. Methods Mol. Biol. 119:187‐194.
   Bornens, M., Paintrand, M., Berges, J., Marty, M.C., and Karsenti, E. 1987. Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskel. 8:238‐249.
   Bornens, M. and Moudjou, M. 1999. Studying the composition and function of centrosomes in vertebrates. Methods Cell Biol. 61:13‐34.
   Evans, L., Mitchison, T., and Kirschner, M. 1985. Influence of the centrosome on the structure of nucleated microtubules. J. Cell Biol. 100:1185‐1191.
   Greenspan, R.J. 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Komesli, S., Tournier, F., Paintrand, M., Margolis, R.L., Job, D., and Bornens, M. 1989. Mass isolation of calf thymus centrosomes: Identification of a specific configuration. J. Cell Biol. 109:2869‐2878.
   Lange, B.M.H. and Gull, K. 1995. A molecular marker for centriole maturation in the mammalian cell cycle. J. Cell Biol. 130:919‐927.
   Lange, B.M.H. and Gull, K. 1996. A structural study of isolated mammalian centrioles using negative staining electron microscopy. J. Struct. Biol. 117:222‐226.
   Lange, B.M.H., 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 J. 19:1252‐1262.
   Mitchison, T.J. and Kirschner, M.W. 1986. Isolation of mammalian centrosomes. Methods Enzymol. 134:261‐268.
   Moritz, M. and Alberts, B.M. 1999. Isolation of centrosomes from Drosophila embryos. Methods Cell Biol. 61:1‐12.
   Moritz, M., Braunfeld, M.B., Fung, J.C., Sedat, J.W., Alberts, B.M., and Agard, D.A. 1995. Three‐dimensional structural characterization of centrosomes from early Drosophila embryos. J. Cell Biol. 130:1149‐1159.
   Palazzo, R.E. and Vogel, J.M. 1999. Isolation of centrosomes from Spisula solidissima oocytes. Methods Cell Biol. 61:35‐56.
   Sullivan, W., Ashburner, M., and Hawley, R.S. (eds.) 2000. Drosophila Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Tavosanis, G., Llamazares, S., Goulielmos, G., and Gonzalez, C. 1997. Essential role for gamma‐tubulin in the acentriolar female meiotic spindle of Drosophila. EMBO J. 16:1809‐1819.
   Wigge, P.A., Jensen, O.N., Holmes, S., Soues, S., Mann, M., and Kilmartin, J.V. 1998. Analysis of the Saccharomyces spindle pole by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. J. Cell Biol. 141:967‐977.
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