Mitotic Spindle Assembly In Vitro

John Merlie1, Rebecca Heald1

1 University of California, Berkeley, Berkeley, Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.13
DOI:  10.1002/0471143030.cb1113s09
Online Posting Date:  May, 2001
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Abstract

The protocols in this unit describe the preparation of materials for an in vitro assay of mitotic spindle assembly in Xenopus egg extracts. Fluorochrome‐labeled tubulin is used to visualize microtubule asters and spindles.

     
 
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Table of Contents

  • Basic Protocol 1: Analysing DMSO and Centrosome Aster Reactions
  • Basic Protocol 2: Analysing Sperm DNA “Half‐Spindle” Reactions
  • Basic Protocol 3: Analysing Sperm DNA “Cycing” Reactions
  • Basic Protocol 4: Analysing DNA‐Bead Reactions
  • Support Protocol 1: Preparation of CSF Extract
  • Support Protocol 2: Preparation of Rhodamine‐Labeled Tubulin
  • Support Protocol 3: Motor Disruption
  • Support Protocol 4: Reaction Spin‐Downs
  • Support Protocol 5: DNA‐Coated Beads
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Analysing DMSO and Centrosome Aster Reactions

  Materials
  • 20 to 30 mg/ml rhodamine‐labeled tubulin (see protocol 6)
  • CSF extract (unit 11.11; also see protocol 5)
  • Dimethyl sulfoxide (DMSO), anhydrous or ∼5 × 108 purified centrosomes/ml
  • Spindle fix (see recipe)
  • Nail polish
  • 1.5‐ml microcentrifuge tubes
  • Wide‐orifice 1‐ to 200‐µl pipet tips
  • Water bath
  • Microscope slides and 18 × 18–mm square coverslips
  • Fluorescence microscope with 40× or 63× lens and rhodamine/Hoechst filter sets

Basic Protocol 2: Analysing Sperm DNA “Half‐Spindle” Reactions

  Materials
  • 20 to 30 mg/ml rhodamine tubulin stock (Murray, )
  • CSF extract (unit 11.11; also see protocol 5)
  • 20× demembranated sperm nuclei (∼100 sperm/µl extract; unit 11.10)
  • Spindle fix (see recipe)
  • Nail polish
  • 1.5‐ml microcentrifuge tubes
  • Water bath
  • Microscope slides and 18 × 18–mm square coverslips
  • Fluorescence microscope with 40× or 63× lens and rhodamine/Hoechst filter sets

Basic Protocol 3: Analysing Sperm DNA “Cycing” Reactions

  Materials
  • 20 to 30 mg/ml rhodamine tubulin stock (see protocol 6)
  • CSF extract (unit 11.11; also see protocol 5)
  • 20× demembranated sperm nuclei (∼100 sperm/µl extract; unit 11.10)
  • 10× calcium solution (see recipe)
  • Spindle fix (see recipe)
  • 20°C water bath
  • Microscope slides and 18 × 18–mm square coverslips
  • Fluorescence microscope with 40× or 63× lens and rhodamine/Hoechst filter sets

Basic Protocol 4: Analysing DNA‐Bead Reactions

  Materials
  • DNA beads (see protocol 9)
  • CSF extract (unit 11.11; also see protocol 5) with and without 1:200 rhodamine‐labeled tubulin ( protocol 6)
  • 10× calcium solution (see recipe)
  • Spindle fix (see recipe)
  • 0.5‐ml microcentrifuge tubes
  • Magnetic particle concentrator (MPC; Dynal)
  • 20°C water bath
  • Microscope slides and 18 × 18–mm square coverslips
  • Fluorescence microscope with 40× or 63× lens and rhodamine/Hoechst filter sets

Support Protocol 1: Preparation of CSF Extract

  Materials
  • Xenopus eggs, dejellied (unit 11.10)
  • Extract buffer (XB; see recipe)
  • CSF‐XB (see recipe)
  • 10 mg/ml LPC (see recipe)
  • 10 mg/ml cytochalasin D in DMSO: store in aliquots at −20°C
  • 20× energy mix (unit 11.11)
  • 400‐ml beaker
  • Polished, cut‐off glass pipet
  • SW‐50 tubes (Beckman)
  • 13‐ml adapter tubes (Sarstedt)
  • Clinical centrifuge
  • Sorvall centrifuge and HB‐4 or HB‐6 rotor with rubber adapters for 15‐ml tubes
  • 18‐G needle and 1‐ml syringe
  • Additional reagents and equipment for injecting frogs, and collecting and dejellying eggs (unit 11.10)

Support Protocol 2: Preparation of Rhodamine‐Labeled Tubulin

  Materials
  • 100 to 200 mg phosphocellulose column–purified tubulin, frozen (Ashford et al., )
  • 20× conversion buffer, ice‐cold (see recipe)
  • 200 mM GTP
  • Glycerol, 37°C
  • Labeling cushion (see recipe)
  • Labeling buffer, 37°C (see recipe)
  • Tetramethylrhodamine succinimidyl ester (Molecular Probes)
  • Dimethyl sulfoxide (DMSO), anhydrous (Sigma)
  • BRB80 cushion, 37°C (see recipe)
  • Quenching buffer, 37°C (see recipe)
  • IB, ice‐cold (see recipe)
  • 2× and 1× BRB80, ice‐cold (see recipe for 5×)
  • 37°C water bath
  • Ultracentrifuge with Ti50 rotor and polycarbonate screw‐cap centrifuge tubes, prewarmed to 35°C (Beckman)
  • 3‐ml wide‐orifice plastic transfer pipets
  • Wash bottle with H 2O
  • TLA100 table‐top ultracentrifuge with TLA100.3 rotor, TL100 rotor, and polycarbonate centrifuge tubes, prewarmed to 35°C (Beckman)
  • Sonicator
  • Additional equipment and reagents for determining protein concentration by Bradford assay ( appendix 3B)

Support Protocol 3: Motor Disruption

  Materials
  • Dynein intermediate chain antibody 70.1 (Sigma), as ascites
  • Unreactive antibody (e.g., mouse IgG or ascites; Sigma)
  • Extract buffer (XB; see recipe)
  • 12,000 to 14,000 mol.‐wt. cut‐off dialysis tubing
  • 4°C microcentrifuge
  • Low‐volume concentrators with mol. wt. 50,000 cut‐off (Microcon)

Support Protocol 4: Reaction Spin‐Downs

  • Extract reaction
  • 30% or 15% spin‐down dilution buffer (see recipe)
  • 40% or 25% spin‐down cushion (see recipe)
  • Methanol, −20°C
  • Wash buffer: PBS, sterile/0.1% (v/v) NP‐40
  • PBS, sterile/3% (w/v) BSA
  • Primary and secondary antibodies in PBS, sterile/3% BSA (e.g., GTU88 anti γ‐tubulin and FITC‐conjugated goat anti‐mouse; Sigma)
  • 10 mg/ml Hoechst dye, in water
  • Mounting medium (see recipe)
  • 1.5‐ml microcentrifuge tube
  • Modified 15‐ml Corex tube containing 12‐mm round coverslip (Aladin Enterprises; see Fig. )
  • Wide‐orifice 200‐ to 1000‐µl pipet tips
  • Sorvall centrifuge, HB‐4, HB‐6, or HS‐4 rotor and appropriate rubber adapters
  • Metal hook or microspatula
  • Watchmaker's forceps
  • Ceramic staining rack
  • Glass staining dish
  • Incubation chamber: parafilm cut to fit in bottom of 150‐cm round plastic tissue culture dish with lid
NOTE: When using this protocol for either type of aster reaction (see protocol 1), substitute 15% spin‐down dilution buffer and 25% spin‐down cushion in place of those called for below.

Support Protocol 5: DNA‐Coated Beads

  Materials
  • 50 µg plasmid DNA (<5 kb) purified by column (Qiagen) chromatography
  • Appropriate restriction enzymes (e.g., NotI, BamHI)
  • TE buffer, pH 8, sterile ( appendix 2A)
  • Klenow DNA polymerase (exo) and buffer (New England Biolabs)
  • Nucleotides: biotin‐dATP (Life Technologies), biotin‐dUTP (Clontech), thio‐dCTP, and thio‐dGTP (Pharmacia)
  • G‐50 Nick columns (Pharmacia)
  • Washing and binding solutions from Kilobase BINDER kit (Dynal)
  • Streptavidin Dynabeads from Kilobase BINDER kit (Dynal)
  • Bead buffer (see recipe)
  • Magnetic particle concentrator (MPC; Dynal)
  • Rotator at 16°C
  • Additional reagents and equipment for ethanol precipitation and restriction endonuclease digestion of plasmid DNA (see appendix 3A), and quantification by absorption spectroscopy ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Andersen, S.S.L. 1999. Balanced regulation of microtubule dynamics during the cell cycle: A contemporary view. BioEssays 21:53‐60.
   Ashford, A.J., Anderson, S.S.L., and Hyman, A.A. 1998. Preparation of tubulin from bovine brain. In Cell Biology: A Laboratory Handbook, 2nd ed. (J.E. Celis, ed.) Vol. 2, pp. 205‐212. Academic Press, San Diego.
   Belmont, L.D., Hyman, A.A., Sawin, K.E., and Mitchison, T.J. 1990. Real‐time visualization of cell cycle‐dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62:579‐589.
   Blomberg‐Wirschell, M. and Doxsey, S.J. 1998. Rapid isolation of centrosomes. Methods Enzymol. 298:228‐238.
   Desai, A., Deacon, H.W., Walczak, C.E., and Mitchison, T.J. 1997. A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts. Proc. Natl. Acad. Sci. U.S.A. 94:12378‐12383.
   Gaglio, T., Saredi, A., Bingham, J.B., Hasbani, M.J., Gill, S.R., Schroer, T.A., and Compton, D.A. 1996. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole. J. Cell Biol. 135:399‐414.
   Gaglio, T., Dionne, M.A., and Compton, D.A. 1997. Mitotic spindle poles are organized by structural and motor proteins in addition to centrosomes. J. Cell Biol. 138:1055‐1066.
   Heald, R. and Walczak, C.E. 1999. Microtubule‐based motor function in mitosis. Curr. Opin. Struct. Biol. 9:268‐274.
   Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A., and Karsenti, E. 1996. Self‐organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382:420‐425.
   Heald, R., Tournebize, R., Habermann, A., Karsenti, E., and Hyman, A. 1997. Spindle assembly in Xenopus egg extracts: Respective roles of centrosomes and microtubule self‐organization. J. Cell Biol. 138:615‐628.
   Hirano, T. and Mitchison, T.J. 1991. Cell cycle control of higher‐order chromatin assembly around naked DNA in vitro. J.Cell Biol. 115:1479‐1489.
   Hyman, A.A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., and Mitchison, T.J. 1991. Preparation of modified tubulins. Methods Enzymol. 196:478‐485.
   Lohka, M.J. and Maller, J.L. 1985. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell‐free extracts. J. Cell Biol. 101:518‐523.
   Lohka, M.J. and Masui, Y. 1983. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220:719‐721.
   Mitchison, T.J. and Kirschner, M.W. 1984. Microtubule assembly nucleated by isolated centrosomes. Nature 312:232‐236.
   Mountain, V. and Compton, D.A. 2000. Dissecting the role of molecular motors in the mitotic spindle. Anat. Rec. 261:14‐24.
   Murray, A.W. 1991. Cell cycle extracts. Methods Cell Biol. 36:581‐605.
   Murray, A.W. and Kirschner, M.W. 1989. Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275‐280.
   Murray, A.W., Desai, A.B., and Salmon, E.D. 1996. Real‐time observation of anaphase in vitro. Proc. Natl. Acad. Sci. U.S.A. 93:12327‐12332.
   Sagata, N., Watanabe, N., Vande, W.G., and Ikawa, Y. 1989. The c‐mos proto‐oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 342:512‐518.
   Sawin, K.E. and Mitchison, T.J. 1991. Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol. 112:925‐940.
   Shamu, C.E. and Murray, A.W. 1992. Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J. Cell Biol. 117:921‐934.
   Verde, F., Labbe, J.C., Doree, M., and Karsenti, E. 1990. Regulation of microtubule dynamics by cdc2 protein kinase in cell‐free extracts of Xenopus eggs. Nature 343:233‐238.
   Verde, F., Berrez, J.M., Antony, C., and Karsenti, E. 1991. Taxol‐induced microtubule asters in mitotic extracts of Xenopus eggs: Requirement for phosphorylated factors and cytoplasmic dynein. J. Cell Biol. 112:1177‐1187.
   Walczak, C.E. 2000. Microtubule dynamics and tubulin interacting proteins. Curr. Opin. Cell Biol. 12:52‐56.
   Walczak, C.E., Vernos, I., Mitchison, T.J., Karsenti, E. and Heald, R. 1998. A model for the proposed roles of different microtubule‐based motor proteins in establishing spindle bipolarity. Curr. Biol. 8:903‐913.
   Zheng, Y., Wong, M.L., Alberts, B., and Mitchison, T. 1995. Nucleation of microtubule assembly by a gamma‐tubulin‐containing ring complex. Nature 378:578‐583.
Key References
   Murray 1991. See above.
  This article documents the Xenopus system and is the basis for many of the protocols presented here.
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