Movement of Nuclei

Sigrid Reinsch1

1 NASA‐Ames Research Center, Moffett Field, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.4
DOI:  10.1002/0471143030.cb1304s10
Online Posting Date:  May, 2001
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Abstract

This unit describes the first assay that reconstructs the movement of the female pronucleus in the newly fertilized frog egg. Nuclei are assembled in frog egg extracts and translocated along microtubules using the microtubule motor dynein.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Nuclear Motility Assay
  • Support Protocol 1: Centrosome Isolation from Lymphocytes
  • Support Protocol 2: Titration of Concentrated Centrosomes
  • Support Protocol 3: Preparing Fractionated Interphase Extracts for Nuclear Assembly
  • Support Protocol 4: Preparation of High‐Speed Supernatant (HSS)
  • Support Protocol 5: Nuclear Assembly Using DNA‐Coated Magnetic Beads as Template
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Nuclear Motility Assay

  Materials
  • Centrosomes (see protocol 2)
  • PE buffer (see recipe)
  • 5 mg/ml casein in PE buffer (see recipe)
  • ABC buffer (see recipe)
  • HSS cytosol (see protocol 5)
  • DNA‐bead nuclei (see protocol 6)
  • Valap (unit 13.1) at 37°C
  • Immersion oil
  • Assay reagent (e.g., drug, antibody, expressed protein)
  • Antibodies bound to beads (for immunodepletion experiments)
  • Inverted microscope equipped for high‐resolution DIC optics
  • Simple perfusion chambers made from: clean slide, double‐stick tape, and clean 18 × 18–mm coverslips (for preparation see unit 13.1, Support Protocol 1)
  • Humid chamber: a covered 10‐ or 15‐cm glass petri dish with strip of moist filter paper around perimeter
  • Magnetic particle concentrator (Dynal)
  • Inverted microscope
  • Camera (e.g., Hamamatsu CCD C307, Hamamatsu)
  • Image processor (e.g., Argus 10, Hamamatsu or equivalent)

Support Protocol 1: Centrosome Isolation from Lymphocytes

  Materials
  • KE37 human lymphoblastic cells (ACC46; DSMZ German Collection of Microorganisms and Cell Cultures)
  • RPMI‐10: RPMI 1640 medium with 10% (w/v) FBS
  • 10 mM nocodozole stock solution in DMSO (see recipe)
  • 10 mg/ml cytochalasin D stock solution in DMSO (see recipe)
  • PBS (see recipe), ice cold
  • PBS/10 with 8% sucrose (see recipe)
  • Lysis buffer (see recipe)
  • 4 ml 0.5 M K‐PIPES (pH 7.2)/1 mM EDTA (see recipe)
  • 1 mg/ml DNase I (see recipe)
  • 40%, 50%, and 70% sucrose in gradient buffer (see recipe,)
  • Liquid nitrogen
  • PE buffer (see recipe)
  • Methanol, −20°C
  • PBS/0.1% Triton X‐100
  • Monoclonal anti‐tubulin antibody (Amersham)
  • Secondary antibodies
  • Hoechst
  • Polyclonal anti‐pericentriolar antibody (e.g., γ‐tubulin, pericentrin; optional)
  • 250‐ml plastic flask
  • 2‐liter spinner culture flask
  • 500‐ml centrifuge bottles
  • Clinical centrifuge or equivalent with swinging bucket rotor for 50‐ml tubes
  • Super‐speed centrifuge (e.g., Sorvall RC‐26 or equivalent)
  • Large volume rotor (GSA or equivalent)
  • 50‐ml capped conical tubes
  • 10‐ml plastic pipet
  • 125‐µm nylon mesh (Millipore)
  • Beckman SW28 and SW28.1 centrifuge tubes
  • Ultracentrifuge
  • Ultracentrifuge SW28 rotor with SW28 and SW28.1 buckets
  • 18‐G needles
  • Refractometer
  • Modified Corex tubes (Evans et al., ; unit 11.13)
  • 11‐ to 12‐mm diameter coverslips (acid‐washed)
  • HB‐4 or HB‐6 rotor
  • Forceps
  • Fixing jar with coverslip holder
  • Additional reagents and equipment for immunofluorescence staining (unit 4.3) and analyzing centrosomes by spinning onto coverslips (unit 11.13)

Support Protocol 2: Titration of Concentrated Centrosomes

  • Large aliquot of centrosomes (see protocol 2)
  • Liquid nitrogen
  • 0.5‐ml microcentrifuge tubes for aliquotting

Support Protocol 3: Preparing Fractionated Interphase Extracts for Nuclear Assembly

  Materials
  • 5 to 7 frogs
  • 100 U pregnant mare serum gonadotropin (PMSG; see recipe)
  • 500 U HCG (see recipe)
  • MMR (see recipe)
  • Dejellying solution (see recipe)
  • S‐lysis buffer (see recipe)
  • S‐lysis‐plus buffer (see recipe)
  • 10 mg/ml cytochalasin D stock solution in DMSO (see recipe)
  • 1 M DTT (see recipe)
  • Protease inhibitors (LPC; see recipe)
  • 2.5 M sucrose (see recipe)
  • Glycerol
  • Liquid nitrogen for freezing aliquots
  • S‐lysis‐plus/500 mM sucrose solution (see recipe)
  • 1‐ml syringes and 27‐G needles
  • 400‐ml beakers
  • SW50 ultraclear centrifuge tubes (Beckman)
  • Pasteur pipet cut to a wide‐mouth bore with a file, fire polished
  • Sarstedt 13‐ml adaptor tubes
  • 2‐ml syringe and 18‐G needle
  • Clinical centrifuge
  • Sorvall RC5 centrifuge
  • HB‐4 rotor with rubber adapters (Sorvall)
  • 5‐ml polyproponate tubes with caps
  • Ultracentrifuge
  • SW55 rotor
  • 500‐ml microcentrifuge tubes for aliquotting cytosol and membranes
NOTE: Make sure that all glassware is clean and rinsed with distilled water before use. Additionally, wet all glassware with buffer before contact with the eggs as they will stick to the glassware and activate or lyse.

Support Protocol 4: Preparation of High‐Speed Supernatant (HSS)

  • ABC buffer (see recipe)
  • Table top ultracentrifuge and TLA‐100 rotor or Airfuge (Beckman) and rotor

Support Protocol 5: Nuclear Assembly Using DNA‐Coated Magnetic Beads as Template

  Materials
  • Cytosol‐200 (see protocol 3)
  • ABC buffer (see recipe)
  • DNA‐Dynabeads (for preparation see unit 11.13, Heald et al., )
  • PBS/1% (w/v) BSA (see recipe)
  • 150 mg/ml glycogen stock (see recipe)
  • M‐200 (see protocol 3)
  • 50 mM Mg‐ATP (see recipe)
  • 0.5 M creatine phosphate (see recipe)
  • 8 mg/ml creatine kinase (see recipe)
  • 1.4 mg/ml TRITC‐BSA‐NLS (transport substrate; unit 11.7, Support Protocol 2)
  • Fix solution (see recipe)
  • Beckman table top ultracentrifuge and TL100 rotor (or Beckman airfuge and rotor)
  • Magnetic particle concentrator
  • Microscope slides and clean coverslips
  • Nail polish
  • Microscope equipped with epifluorescence optics
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Figures

Videos

Literature Cited

Literature Cited
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   Buendia, B., Draetta, G., and Karsenti, E. 1992. Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: Role of cyclin A‐associated protein kinase. J. Cell Biol. 116:1431‐1442.
   Euteneuer, U. and Schliwa, M. 1985. Evidence for an involvement of actin in the positioning and motility of centrosomes. J. Cell Biol. 101:96‐103.
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   Gonczy, P., Pichler, S., Kirkham, M., and Hyman, A.A. 1999a. Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J. Cell Biol. 147:135‐150.
   Gonczy, P., Schnabel, H., Kaletta, T., Amores, A.D., Hyman, T., and Schnabel, R. 1999b. Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis. J. Cell Biol. 144:927‐946.
   Hartl, P., Olson, E., Dang, T., and Forbes, D.J. 1994. Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: An unexpected role for glycogen in formation of a higher‐order chromatin intermediate. J. Cell Biol. 124:235‐248.
   Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, H., and Karsenti, E. 1996. Self‐organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts [see comments]. Nature 382:420‐425.
   Heald, R., Tournebize, R., Vernos, I., Murray, A., Hyman, T., and Karsenti, E. 1998. In vitro assays for mitotic spindle assembly and function. In Cell Biology. A Laboratory Handbook, Vol. 2. (J.E. Celis, ed.) pp. 326‐335. Academic Press, San Diego.
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   Merdes, A., Ramyar, K., Vechio, J.D., and Cleveland, D.W. 1996. A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. Cell 87:447‐458.
   Moudjou, M. and Bornens, M. 1998. Method of centrosome isolation from cultured animal cells. In Cell Biology: A Laboratory Handbook, Vol. 2. (J.E. Celis, ed.) pp. 111‐119. Academic Press, San Diego.
   Murray, A.W. 1991. Cell cycle extracts. In Xenopus laevis: Practical Uses in Cell and Molecular Biology, Vol. 36, Methods in Cell Biology (B.K. Kay and H.B. Peng, eds.) pp. 581‐604. Academic Press, San Diego.
   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.
   Newmeyer, D.D. and Wilson, K.L. 1991. Egg extracts for nuclear import and nuclear assembly reactions. In Xenopus laevis: Practical Uses in Cell and Molecular Biology, Vol. 36, Methods in Cell Biology (B.K. Kay and H.B. Peng, eds.) pp. 607‐634. Academic Press, San Diego.
   Newmeyer, D.D., Farschon, D.M., and Reed, J.C. 1994. Cell‐free apoptosis in Xenopus egg extracts: inhibition by Bcl‐2 and requirement for an organelle fraction enriched in mitochondria. Cell 79:353‐364.
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   Reinsch, S. and Gonczy, P. 1998. Mechanisms of nuclear positioning. J. Cell Sci. 111:2283‐2295.
   Reinsch, S. and Karsenti, E. 1997. Movement of nuclei along microtubules in Xenopus egg extracts. Curr. Biol. 7:211‐214.
   Rouviere, C., Houliston, E., Carre, D., Chang, P., and Sardet, C. 1994. Characteristics of pronuclear migration in Beroe ovata. Cell Motil. Cytoskeleton 29:301‐311.
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   Tournebize, R., Popov, A., Kinoshita, K., Ashford, A.J., Rybina, S., Poznikovsky, A., Mayer, T.U., Walczak, C.E., Karsenti, E., and Hyaman, A.A. 2000. Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts. Nat. Cell Biol. 2:13‐19.
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Key References
   Reinsch and Gonczy 1998. See above.
  This review describes the different mechanisms that drive microtubule‐mediated nuclear motility events in different cell types and different organisms. Nuclear motility following fertilization is particularly emphasized.
   Reinsch and Karsenti 1997. See above.
  This work demonstrates that nuclei can translocate along microtubules in Xenopus egg extracts similar to other organelles, and that cytoplasmic dynein cytoplasmic dynein drives the translocation of nuclei along microtubules.
Internet Resources
  http://current-biology.com/supmat/cub/bb7325s1.mov
  These movies are supplemental material for Reinsch and Karsenti (). They are videos of nuclei moving along microtubule asters as described in the Basic Protocol. The nuclei used in these movies were assembled as described in Support Protocols and 5.
  http://current-biology.com/supmat/cub/bb7325s2.mov
  This is the webpage for the laboratory of Susan Strome at Indiana University. Movies within this site show pronuclear migration in vivo in the nematode C. elegans.
  http://current-biology.com/supmat/cub/bb7325s3.mov
  http://www.indiana.edu/~elegans/
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