Nuclear Import in Digitonin‐Permeabilized Cells

Mary Shannon Moore1, Eric D. Schwoebel1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.7
DOI:  10.1002/0471143030.cb1107s05
Online Posting Date:  May, 2001
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Abstract

Import of proteins into the nucleus of a cell is a complex process that can be reconstituted in vitro. Digitonin‐permeabilized cells are washed free of cytosolic factors to provide competent nuclei. Cytosolic factors for import are provided by an extract of Xenopus ovarian cells. Fluorochrome‐conjugated probes, cloned proteins fused to green fluorescent protein, or antibodies to the protein of interest are used to visualize nuclear import. The system can also be used to identify nuclear localization sequences (NLS) of imported proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Nuclear Import Assay in Attached HeLa Cells
  • Support Protocol 1: Preparation of Xenopus Ovarian Cytosol
  • Support Protocol 2: Production of Fluorescent Import Substrate: TRITC‐BSA‐NLS
  • Support Protocol 3: Production of Fluorescent Recombinant Import Substrate: GFP‐GST‐NLS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Nuclear Import Assay in Attached HeLa Cells

  Materials
  • 70% ethanol
  • 30,000 cell/ml suspension of HeLa cells (ATCC CCL‐2) in complete DMEM/10% FBS
  • DMEM/10% FBS (see recipe)
  • 1× transport buffer (TB; see recipe), ice cold
  • Digitonin working solution (see recipe), ice cold
  • Import reaction mixture (see recipe)
  • Paraformaldehyde working solution (see recipe), ice cold
  • p‐phenylendiamine mounting medium (see recipe)
  • Clear nail polish
  • 12‐mm circular glass coverslips (Fisher) sterilized by autoclaving
  • 24‐well tissue culture plates
  • Whatman 3 mm filter paper
  • Fine‐tipped forceps with large radius curved shanks (e.g., Dumont #7, Fine Science Tools)
  • Fluorescence microscope
NOTE: All solutions and equipment coming into contact with tissue culture cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Support Protocol 1: Preparation of Xenopus Ovarian Cytosol

  Materials
  • Eight female Xenopus frogs
  • PBS (see recipe), ice‐cold
  • Protease inhibitor tablets (Boehringer Mannheim Complete, EDTA‐free)
  • 1× homogenization buffer (see recipe), ice cold
  • 1× transport buffer (TB; see recipe), ice cold
  • Carbon dioxide tank with attached tubing
  • Polytron homogenizer, PRO300D (PRO Scientific)
  • 250‐ml plastic centrifuge bottles
  • Sorvall centrifuge with GSA centrifuge rotor (or equivalent)
  • Cheesecloth
  • Beckman ultracentrifuge with Ti 70.1 rotor (or equivalent), and appropriate ultracentrifuge tubes
  • Plastic tubing, thin
  • 20‐ml syringe
  • Dialysis tubing (4000 to 6000 MWCO)
  • Centriplus‐10 concentrators (Amicon)
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)

Support Protocol 2: Production of Fluorescent Import Substrate: TRITC‐BSA‐NLS

  Materials
  • Sephadex G‐25 and G‐50 (Pharmacia Biotech)
  • 0.1 M sodium phosphate, pH 7.0 and 8.0 ( appendix 2A), ice cold
  • Bovine serum albumin, fatty‐acid free (Boehringer Mannheim)
  • 0.1 M sodium carbonate, pH 9.0/50 mM NaCl (store indefinitely at room temperature)
  • Tetramethylrhodamine‐5‐(and 6)‐isothiocyanate (TRITC; Molecular Probes)
  • DMSO
  • 1.5 M hydroxylamine⋅HCl, pH 8.5 (see recipe)
  • Sulfo‐SMCC (Pierce)
  • NLS or mutant NLS peptide (∼1 mg)
  • 20 mM HEPES (potassium salt), pH 7.3/110 mM potassium acetate (prepare fresh)
  • 1× transport buffer (TB; see recipe)
  • Disposable 20‐ml columns (Bio‐Rad Laboratories, Cat # 732‐1010)
  • Rocking platform
  • Centricon‐30 concentrators (Amicon)
  • Additional reagents and equipment for dialysis ( appendix 3C), determining protein concentration ( appendix 3B), and SDS‐PAGE (unit 6.1)

Support Protocol 3: Production of Fluorescent Recombinant Import Substrate: GFP‐GST‐NLS

  Materials
  • LB medium ( appendix 2A) containing 100 µg/ml ampicillin
  • DH5α strain of E. coli
  • GST‐GFP‐NLS and GST‐GFP‐mutNLS constructs in E. coli (Dr. Manfred Lohka, Biological Sciences, University of Calgary, Calgary, Canada)
  • 1 M IPTG in H 2O, sterile filtered
  • Phosphate‐buffered saline (PBS; see recipe)
  • Protease inhibitors tablets (Boehringer Mannheim Complete, EDTA‐free)
  • PBS/EDTA (see recipe)
  • 10% (v/v) Triton X‐100
  • Glutathione–Sepharose 4B (Pharmacia Biotech)
  • Glutathione/Tris (see recipe)
  • 20 mM HEPES (potassium salt), pH 7.3/110 mM potassium acetate (prepare fresh)
  • Transport buffer (TB; see recipe)
  • Shaking incubator
  • Sorvall centrifuge with GSA and SS‐34 rotors (or equivalents)
  • 50‐ml conical centrifuge tubes
  • French press or sonicator
  • Rocking platform
  • Disposable 2‐ml column (Bio‐Rad)
  • Centricon‐30 (Amicon)
  • Additional reagents and equipment for dialysis ( appendix 3B), determining protein concentration ( appendix 3B), and SDS‐PAGE (unit 6.1)
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Figures

Videos

Literature Cited

Literature Cited
   Adam, S.A. 1998. Permeabilized cell assay for nuclear protein import. In Cells, A Laboratory Manual, Vol. 1: Culture and Biochemical Analysis of Cells (D.L. Spector, R.D. Goldman, and L.A. Leinwand, eds.) pp. 45.1‐45.8. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Adam, E.J.H. and Adam, S.A. 1994. Identification of cytosolic factors required for nuclear location sequence‐mediated binding to the nuclear envelope. J. Cell Biol. 125:547‐555.
   Adam, S.A. and Gerace, L. 1991. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66:837‐847.
   Adam, S.A., Lobl, T.J., Mitchell, M.A., and Gerace, L. 1989. Identification of specific binding proteins for a nuclear location sequence. Nature 337:276‐279.
   Adam, S.A., Sterne‐Marr, R., and Gerace, L. 1990. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111:807‐816.
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   Bonner, W.M. 1975. Protein migration into nuclei. I. Frog nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. J. Cell Biol. 64:421‐430.
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   Moore, M.S. and Blobel, G. 1992. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell 69:939‐950.
   Moore, M.S. and Blobel, G. 1993. The GTP‐binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365:661‐663.
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