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Analysis of Nuclear Protein Import and Export in Digitonin-Permeabilized Cells
|Analysis of Nuclear Protein Import and
Export in Digitonin-Permeabilized Cells
Transport between the nucleus and the cytoplasm is
mediated by nuclear pore complexes (NPCs), specialized
channels that are embedded in the nuclear envelope
membrane. Proteins that undergo nuclear import
or nuclear export usually encode a nuclear localization
signal (NLS) or a nuclear export signal (NES). These
signals are recognized by import or export receptors
that, in turn, facilitate targeting of the protein to the
NPC and translocation through the central channel of
the NPC. Both import and export pathways are regulated
by the Ras-related GTPase Ran. Nuclear Ran
in its GTP-bound form promotes the release of NLSproteins
from import receptors after the NLSprotein/
import receptor has reached the nuclear side
of the NPC. In contrast, nuclear Ran in its GTP-bound
form promotes the assembly of NES-containing proteins
with export receptors by forming an NESprotein/
export receptor/RanGTP. Export complexes
are then disassembled on the cytoplasmic side of the
NPC through the action of a Ran GTPase-activating
protein that stimulates GTP hydrolysis by Ran. More
specific information regarding import and export
complex assembly, models for translocation, and additional
aspects of nuclear transport regulation are
described elsewhere (Steggerda and Paschal, 2002;
Weis, 2003 and references therein).
Digitonin-permeabilized cells have become one of
the most widely used experimental systems for studying
nuclear transport (Adam et al.
, 1990). They have
been used to analyze nuclear import and export
signals (Pollard et al.
, 1996), to purify nuclear transport
factors from cell extracts (Görlich, 1994; Paschal and
Gerace, 1995; Kehlenbach et al.
, 1998), and to measure
nuclear transport kinetics
(Görlich and Ribbeck, 2003).
In all of these applications, the principle is that treating
mammalian cells with a defined concentration of
digitonin results in selective perforation of the plasma
membrane, leaving the nuclear membrane intact.
Thus, soluble transport factors are released from the
cytoplasmic compartment and low molecular weight
transport factors such as Ran (24 kDa) and NTF2
(28 kDa) are released from the nuclear compartment
by diffusion through the NPC. Nuclear import and
export can subsequently be reconstituted in digitoninpermeabilized
cells by the addition of transport
factors, an energy-regenerating system, and inclusion
of a fluorescent NLS or NES reporter protein to
monitor transport (Adam et al.
, 1990). Transport factors
can be supplied as unfractionated cytosol from HeLa
cells or as commercially available rabbit reticulocyte
lysate. The level of import is then measured by following
the accumulation of an NLS reporter in the
nucleus or, in the case of export, by the loss of an NES
reporter from the nucleus.
This article describes assays for both nuclear import
and export (Fig. 1). The nuclear import assay can be
performed with various cell lines and analyzed by fluorescence
microscopy. The nuclear export assay is
described for HeLa cells expressing a GFP-tagged
reporter protein, the nuclear factor of activated T cells
(GFP-NFAT). NFAT is a transcription factor that contains
defined nuclear localization and nuclear export
signals and that shuttles between the cytoplasm and
the nucleus in a phosphorylation-dependent manner
(for review, see Crabtree and Olsen, 2002). We take
advantage of the tight regulation of nucleocytoplasmic
transport of GFP-NFAT to induce nuclear import in
intact cells, followed by export under controlled conditions
from nuclei of permeabilized cells (Kehlenbach et al.
, 1998). The efficiency of nuclear export can be analyzed
either qualitatively by fluorescence microscopy
(see Fig. 3) or quantitatively by flow cytometry (see
Fig. 4). In the latter case, nuclear import of a fluorescently
labeled reporter protein can be analyzed in
II. MATERIALS AND
|FIGURE 1 Overview of assays for measuring nuclear import and export in digitonin-permeabilized cells.
See text for details.
Rabbit recticulocyte lysate (L4151) is from Promega.
HeLa S3 cells (CCL-2.2) and NIH 3T3 cells are from
the American Type Culture Collection. Joklik's
modified minimum essential medium for suspension
cell culture (JMEM; Cat. No. M0518) is from
Sigma. Newborn calf serum (Cat. No. 16010-159),
penicillin-streptomycin (Cat. No. 15140-122), and
trypsin EDTA (Cat. No. 25200-056) are from
GIBCO/Invitrogen. ATP (Cat. No. A-2383), creatine
phosphate (Cat. No. P-7936), creatine phosphokinase
(Cat. No. C-7886), sodium bicarbonate (Cat. No. S-
4019), HEPES (Cat. No. H-7523), potassium acetate
(Cat. No. P-5708), magnesium acetate (Cat. No. M-
2545), EGTA (Cat. No. E-4378), dithiothreitol (DTT,
Cat. No. D-5545), NaCl (Cat. No. S-9763), potassium
phosphate, monobasic (Cat. No. P-0662), phenyl-methylsulfonyl fluoride (PMSF, Cat. No. P-7626),
dimethyl sulfoxide (DMSO, Cat. No. D-8779), sodium
carbonate (Cat. No. S-7795), trypan blue (Cat. No.
T-8154), ionomycin (Cat. No. 1-0634), LiOAc (Cat.
No. L-4158), Hoechst 33258 (Cat. No. B-2883),
4',6-diamidino-2-phenyindole, dilactate (DAPI; Cat.
No. 9564), and trichostatin A (Cat. No. T-8552) are from
Sigma. High-purity bovine serum albumin (BSA, Cat.
No. 238031), aprotinin (Cat. No. 236624), leupeptin
(Cat. No. 1017101), and pepstatin (Cat. No. 253286) are
from Roche. The FITC isomer I (Cat. No. F-1906) and
sulfo-SMCC (Cat. No. 22322) are from Molecular
Probes and Pierce, respectively. PD-10 columns (Cat.
No. 17-0851-01), Cy2 (Cat. No. PA-22000), and Cy5
(Cat. No. PA-25001) are from Amersham Pharmacia
Biosciences. Centricon filters (30-kDa cutoff, Cat. No.
4208) are from Amicon. High-purity digitonin (Cat.
No. 300410) is from Calbiochem. Vectashield mounting
medium (Cat. No. H-1000) is from Vector Laboratories.
Six-well dishes (Cat. No. 3516) are from Corning. Glass
coverslips (#1 thickness; Cat. No. 12-548A) and the
polystyrene tubes used for the transport assays (Cat.
No. 2058) are from Fisher. The microscope used for
measuring nuclear import in adherent cells is a Nikon
E800 equipped with a charge-coupled device camera.
The system is linked to a Macintosh computer running
OpenLab software for image acquisition. An Olympus
IX70 inverted fluorescence microscope is used for
analysis of nuclear export. Images from import and
export assays are processed using Adobe Photoshop.
The flow cytometric analysis system is the FACScan
unit from Beckton-Dickinson.
The following equipment required for growing and
harvesting HeLa cells is from Bellco Glass. The 250-ml
spinner flask (Cat. No. 1965-00250) and stir plate (Cat.
No. 7760-0600) are used for the continuous culture of
HeLa cells. Additional spinner flasks are required to
scale up the preparation to 15 liters. These include
100 ml (Cat. No. 1965-01000), 3000 ml (Cat. No. 1965-
03000), and 15 liter (Cat. No. 7764-00110), cap assembly
(Cat. No. 7764-10100), and Teflon paddle assembly
(Cat. No. 1964-30015).
Equipment for centrifugation includes the JS5.2
swinging bucket rotor and J6B centrifuge, the JA-20
fixed angle rotor and J2 centrifuge, and the type 60Ti
fixed angle rotor and L7 centrifuge, all from Beckman.
Cytosol dialysis is carried out using the collodion
vacuum dialysis apparatus (Cat. No. 253310), and
10,000-Da cutoff membranes (Cat. No. 27110) are from
Schleicher and Schuell. The homogenizer used for cell
disruption is a 0.02-mm-clearance stainless-steel unit
(Cat. No. 885310-0015) from Kontes.
A. Preparation of Cytosol
The rabbit reticulocyte lysate contains all the soluble
factors necessary to reconstitute import and export in
digitonin-permeabilized cells (Adam et al.
, 1990). The
only preparation involved is dialysis against 1× transport
buffer containing 2mM
DTT and 1 µg/ml each
of aprotinin, leupeptin, and pepstatin (two buffer
changes). Reticulocyte lysate is used at 50% (by
volume) of transport reactions. This results in a relatively
high total protein concentration (~25-40 mg/ml)
in transport assays because the reticulocyte lysate contains
a high concentration of hemoglobin. A more
economical approach, especially if larger quantities
of cytosol are needed for protein purification, is to
prepare cytosol from suspension culture HeLa cells. In
this case, only 1-2mg/ml (final assay concentration) of
HeLa cell cytosol is required to obtain a maximum
level of protein import or protein export in digitoninpermeabilized
- 10× transport buffer: 200mM HEPES, pH 7.4, 1.1 M potassium acetate, 20mM magnesium acetate, and
5 mM EGTA. To make 1 liter, dissolve 47.6g HEPES,
107.9g potassium acetate, 4.3g magnesium acetate,
and 1.9 g EGTA in 800 ml distilled water. Adjust the pH
to 7.4 with 10 N NaOH and bring the final volume to
1 liter. Sterile filter and store at 4°C.
- 1× transport buffer: 20mM HEPES, pH 7.4, 110M potassium acetate, 2mM magnesium acetate, and
0.5 mM EGTA. To make 1 liter, add 100ml of 10x transport
buffer to 900ml distilled water.
- 1M HEPES stock, pH 7.4: To make 500 ml, dissolve
119.1 g HEPES (free acid) in 400ml distilled water.
Adjust the pH to 7.4 with 10 N NaOH and bring the
final volume to 500ml. Sterile filter and store at 4°C.
- 1M potassium acetate stock: To make 500ml, dissolve
49 g potassium acetate in 400ml distilled water.
Bring the final volume to 500 ml, sterile filter, and store
- 1M magnesium acetate stock: To make 500 ml, dissolve
107.2g magnesium acetate (tetrahydrate) in
400 ml distilled water. Bring the final volume to 500 ml,
sterile filter, and store at 4°C.
- 0.2M EGTA stock: To make 500ml, dissolve 38g
EGTA (free acid) in 400ml distilled water. Adjust the
pH to -7.0 with 10 N NaOH and bring the final volume
to 500ml. Store at 4°C.
- Cell lysis buffer: 5mM HEPES, pH 7.4, 10mM potassium acetate, 2mM magnesium acetate, and 1 mM EGTA. To make 500ml, combine 2.5ml 1M HEPES, pH 7.4, 5 ml 1M potassium acetate, 1 ml 1M magnesium acetate, 2.5 ml 0.2M EGTA, and 489ml distilled
water. Store at 4°C.
- Phosphate-buffered saline (PBS): To make 1 liter,
dissolve 8 g sodium chloride, 0.2 g potassium chloride,
1.44g sodium phosphate (dibasic), and 0.24g potassium
phosphate (monobasic) in 900ml distilled water.
Adjust the pH to 7.4 and bring the final volume to
1 liter. Store at 4°C.
B. Preparation of FITC-BSA-NLS Import
- Grow HeLa cells at a density of 2-7 × 105 cells per
milliliter in a spinner flask (30-50rpm) in a 37°C incubator
(CO2 is not required). The medium is JMEM
containing 2.0 g sodium bicarbonate and 2.38 g HEPES
per liter. Adjust the pH to 7.3, sterile filter, and store at
4°C. Before use, supplement the medium with 10%
newborn calf serum and 1% penicillin-streptomycin.
The cells should have a doubling time of approximately
18 h, making it necessary to dilute the culture
with fresh, prewarmed medium every 1-2 days.
- HeLa cells from a 250-ml culture provide the
starting point for scaling up the preparation to 15
liters. This is carried out by sequential dilution of the
culture into larger spinner flasks. The culture should
not be diluted to a density below 2 × 105 cells/ml. The
spinner flasks used for scaling up preparation are
250ml (1 each), 1 liter (1 each), 3 liters (2 each), and
15 liters (1 each). This process generally takes 5 days.
- Perform the cell harvest and subsequent steps at
0-4°C. Collect the cells by centrifugation (300g for
15 min) in 780-ml conical glass bottles in a Beckman J6B
refrigerated centrifuge equipped with a JS5.2 swinging
bucket rotor. The cell harvest takes about 1 h.
- Wash the cells by sequential resuspension and
centrifugation. Two washes are carried out in ice-cold
PBS (1 liter each) and one wash is carried out in 1× transport buffer containing 2 mM DTT. The yield from
a 15-liter culture should be approximately 40ml of
- Resuspend the cell pellet using 1.5 volume of
lysis buffer, supplemented with 3µg/ml each aprotinin,
leupeptin, pepstatin, 0.5mM PMSF, and 5mM DTT. Allow the cells to swell on ice for 10 min.
- Disrupt the cells by two to three passes in a
stainless-steel homogenizer. Monitor the progress of
homogenization by trypan blue staining and phasecontrast
microscopy. The goal is to obtain -95% cell
disruption. Excessive homogenization should be
avoided because it results in nuclear fragmentation
and the release of nuclear contents into the soluble
fraction of the preparation.
- Dilute the homogenate with 0.1 volume of 10x
transport buffer and centrifuge in a fixed angle rotor
such as the Beckman JA-20 (40,000g for 30min).
- Filter the resulting low-speed supernatant fraction
through four layers of cheesecloth. Subject the
filtered low-speed supernatant to ultracentrifugation
using a fixed angle rotor such as the Beckman type 60
Ti (150,000 g for 60 min).
- Dispense the resulting high-speed supernatant
fraction (-50ml, protein concentration-5mg/ml) into
1- and 4-ml aliquots, flash freeze in liquid N2, and store
at -80°C indefinitely.
- HeLa cell cytosol is generally subjected to a
rapid dialysis step before use in transport reactions.
Thaw a 4-ml aliquot of cytosol at 0-4°C and dialyze for
3 h in 1× transport buffer containing 2mM DTT and
1 µg/ml each of aprotinin, leupeptin, and pepstatin
(two buffer changes). We use a vacuum apparatus and
a collodion membrane to achieve a twofold concentration
of the sample (10mg/ml). Dispense the dialyzed,
concentrated cytosol into 100-µl aliquots, flash freeze
in liquid N2, and store at -80°C.
Synthetic peptides containing an NLS can be used
to direct the nuclear import of a variety of fluorescent
reporter proteins. FITC- or Cy2-BSA-NLS and Cy5-
BSA-NLS are all suitable for measuring import in the
fluorescence microscope or in the flow cytometer.
Because excitation and emission spectra of Cy5 are distinct
from GFP, Cy5-BSA-NLS is ideal for measuring
import and GFP-NFAT export in the same cells. Importantly,
the average size of the protein conjugate
(>70kDa) is too large to allow diffusion through the
NPC and it displays low nonspecific binding to the
permeabilized cell. Preparation of fluorescent import
ligands is carried out in three steps: fluorescent labeling
of BSA, modification of the fluorescent BSA with
the heterobifunctional cross-linker sulfo-SMCC, and
attachment of NLS peptides. Sulfo-SMCC provides a
covalent linkage between primary amines on BSA and
cysteine present on the N terminus of the NLS peptide.
- PBS: To make 1 liter, dissolve 8g sodium chloride,
0.2g potassium chloride, 1.44g sodium phosphate
(dibasic), and 0.24g potassium phosphate
(monobasic) in 900ml distilled water. Adjust the pH
to 7.4 and bring the final volume to 1 liter. Store at
- 0.1M sodium carbonate: To prepare 250ml, dissolve
3.1 g sodium carbonate in 200ml distilled water,
adjust the pH to 9.0, and bring the final volume to
250ml. Store at 4°C.
- 1.5 M hydroxylamine: To prepare 100 ml, dissolve
10.4 g in a total volume of 100ml distilled water. Store
at room temperature.
- 10 mg/ml FITC: Add 1 ml DMSO to 10 mg FITC in
an amber vial and vortex to dissolve.
- 20mM sulfo-SMCC: Prepare a 20mM stock of
sulfo-SMCC in the following manner. Preweigh a
microfuge tube on a fine balance and use a small
spatula to add approximately 1-2mg of sulfo-SMCC
to the microfuge tube. Reweigh the tube containing the
sulfo-SMCC and add DMSO for a final concentration
C. Preparation of Cy2- or Cy5-BSA-NLS
- Dissolve 10mg high-purity BSA in 1 ml sodium
carbonate buffer, pH 9.0.
- Stir the BSA solution in a glass test tube with a
microstir bar and add 0.1 ml of 10mg/ml FITC. Cover
with foil and stir for 60min at room temperature.
- Stop the reaction by adding 0.1ml 1.5M
- Separate FITC-labeled BSA from unincorporated
FITC by desalting on a PD-10 column equilibrated in
PBS, collecting 0.5-ml fractions. The bright yellow
FITC-BSA will elute in the void volume of this column.
- Pool the four or five most concentrated fractions,
dispense into 1-mg aliquots, and freeze in foilwrapped
microfuge tubes at -20°C.
- Combine 1 mg of FITC-BSA with 50 µl of freshly
prepared 20 mM sulfo-SMCC and mix end over end for
45 min at room temperature.
- Separate the sulfo-SMCC-activated FITC-BSA
from unincorporated sulfo-SMCC by desalting on a
PD-10 column equilibrated in PBS. After loading the
sample, fill the buffer reservoir of the column with PBS
and collect 0.5-ml fractions. The bright yellow
FITC-BSA will elute in the void volume as before.
- Pool the three most concentrated fractions of
sulfo-SMCC-activated FITC-BSA and combine with
0.3 mg of NLS peptide (CGGGPKKKRKVED). Mix end
over end in a foil-wrapped microfuge tube overnight
- Remove unincorporated NLS peptide by subjecting
the sample to four cycles of centrifugation and
resuspension in 1× transport buffer using a 2-ml
30-kDa cutoff Centricon filter. Follow the manufacturer's
recommendations for centrifugation
- Adjust the FITC-BSA-NLS conjugate to a final
concentration of 2 mg/ml, dispense into 50-µl aliquots,
flash freeze in liquid N2, and store at -80°C.
The preparation of Cy2- and Cy5-1abeled import
substrate is very similar to the method described
earlier for the FITC-labeled substrate.
D. Nuclear Protein Import Assay
- Dissolve 2.5mg BSA in 1 ml 0.1M sodium carbonate.
Use one vial-activated Cy2 or Cy5 for coupling.
Incubate for 40min at room temperature.
- Separate the CyDye-BSA conjugate from the free
dye by chromatography on a PD-10 column equilibrated
- To activate CyDye-BSA, add sulfo-SMCC to a
final concentration of 2mM. Incubate for 30min at
room temperature. Remove free cross-linker using a
PD-10 column as described earlier.
- Dissolve 1 mg of NLS-peptide (CGGGPKKKRKVED)
with activated CyDye-BSA and incubate
the solution overnight at 4°C. Remove free peptide
and adjust protein concentration as described
- Freeze aliquots in liquid nitrogen and store at
-80°C. After thawing, the import substrate can be kept
at 4°C in the dark for a few weeks.
- 10× and 1 × transport buffer: See Section III,A.
- Complete transport buffer: 1× transport buffer containing
1 µg/ml each aprotinin, leupeptin, pepstatin,
and 2 mM DTT.
- 10% digitonin: To make 2ml, add 0.2g highpurity
digitonin to 1.7ml DMSO and dissolve by
vigorous vortexing, Dispense into 20-µl aliquots and
freeze at -20°C.
- 100mM MgATP: To make 5ml, add 0.5ml 1M magnesium acetate and 0.1 ml 1M HEPES, pH 7.4, to
4ml distilled water. Add 275.1mg ATP, dissolve by
vortexing, and bring the final volume to 5 ml. Dispense
into 20-µl aliquots and freeze at -80°C.
- 250mM creatine phosphate: To make 5ml, add
0.32 g creatine phosphate to 4ml distilled water, dissolve
by vortexing, and bring the final volume to 5 ml.
Dispense into 20-µl aliquots and freeze at -20°C.
- 2000 U/ml creatine phosphokinase: To make 5ml,
dissolve 10,000U creatine phosphokinase in 20mM HEPES, pH 7.4, containing 50% glycerol. Dispense into
1-ml aliquots and store at -20°C.
E. Nuclear Protein Export Assay
Solutions and Reagents
- Plate NIH 3T3 cells onto glass coverslips in sixwell
dishes at a density of ~5 × 104 cells/well and grow
- Place the six-well dishes on ice. Aspirate media
and gently replace with 2 ml ice-cold, complete transport
buffer. Aspirate the transport buffer and replace
with fresh transport buffer twice, taking care not to
disturb the cells. Complete transport buffer in this
and subsequent steps refers to ice-cold, 1× transport
buffer supplemented with DTT and protease
- To permeabilize the cells, aspirate the transport
buffer from each well and immediately add 0.05% digitonin
diluted into complete transport buffer. Incubate
for 5 min on ice.
- Stop the permeabilization reaction by aspirating
the digitonin solution and replacing with complete
transport buffer. Wash the cells twice by alternate steps
of aspiration and buffer addition.
- Assemble the import reactions in 0.6-ml
microfuge tubes on ice. Each reaction contains (final
concentration given) unlabeled BSA (5mg/ml), FITCBSA-
NLS (25 µg/ml), MgATP (1 mM), MgGTP (1 mM),
creatine phosphate (5mM), creatine phosphpkinase
(20U/ml), rabbit reticulocyte lysate (25µl), and complete
transport buffer in a total volume of 50 µl.
- Create an incubation chamber by lining a fiatbottomed,
air-tight box with parafilm and include a
moistened paper towel in the chamber as a source of
humidity. Place the chamber on ice.
- Using fine forceps, remove each coverslip, wick
excess buffer using filter paper, and place cells side up
on the parafilm. Pipette the import reaction onto the
coverslip surface without introducing bubbles.
- Float the incubation chamber on a 30°C water
bath for 20 min.
- Using fine forceps, remove each coverslip, wick
most of the import reaction using filter paper, and
immediately place back into the wells of the six-well
- Wash the coverslips twice by alternate steps of
aspiration and complete transport buffer addition.
- Fix the coverslips by aspirating the complete
transport buffer, adding formaldehyde (3.7%)
diluted into PBS, and incubating for 15min at room
- Remove each coverslip, submerge in distilled
water briefly, wick excess water, and mount on glass
slides using Vectashield. Seal the edges with clear nail
- View the cells by fluorescence microscopy, and
quantify the nuclear fluorescence in 50-100 cells per
condition using image analysis software such as
- The HeLa cell line stably expressing GFP-NFAT,
which is used for the export assay, has been described
in detail (Kehlenbach et al., 1998) and is available upon
- 1 mM trichostatin A: Dissolve 1 mg trichostatin A
in 3.3ml ethanol. Store in aliquots at -20°C. Trichostatin
A is an inhibitor of histone deacetylases and
promotes the expression of GFP-NFAT.
- 1mM ionomycin: Dissolve 1 mg ionomycin in
1.34ml DMSO. Store in aliquots at -20°C. Nuclear
accumulation of the reporter protein is induced by the
calcium ionophore ionomycin.
- Double-stranded oligonucleotides: Dissolve
and 5'TATGAAACAAATTTTCCTCT), each at 200 µM,
in 40 mM Tris, pH 7.4, 20 mM MgCl2, and 50 mM NaCl.
Anneal by heating to 65°C for 5 min and slow cooling
to room temperature. Freeze in aliquots and store at
-20°C. The oligonucleotide sequence corresponds to a
DNA-binding site of NFAT. It stimulates export of
GFP-NFAT about twofold, probably by releasing the
protein from chromatin.
- 1× transport buffer with LiOAc: 20mM HEPES, pH
7.4, 80mM potassium acetate, 2mM magnesium
acetate, and 0.5mM EGTA. To make 1 liter, dissolve
4.76g HEPES, 7.85g potassium acetate, 3.06g lithium
acetate dihydrate, 0.43g magnesium acetate tetrahydrate,
and 0.19 g EGTA in 800 ml distilled water. Adjust
the pH to 7.4 with 1 N KOH and bring the final volume
to 1 liter. Before use, add 1µg/ml each of aprotinin,
leupeptin, pepstatin, and 2mM DTT.
- Complete transport buffer: See Section III, D.
- To stimulate expression of GFP-NFAT, add
250nM trichostatin A to stably transfected HeLa cells
and incubate overnight. One 15-cm dish containing
~106 cells is sufficient for 30 reactions.
- Induce nuclear import of GFP-NFAT by adding
1 µM ionomycin and 30mM LiOAc directly to the
culture media and return the cells to the 37°C incubator for 30min. Lithium inhibits one of the kinases involved in nuclear phosphorylation of NFAT, a step
that is required for efficient export in vivo.
- Rinse cells with PBS and remove from dish by
adding trypsin EDTA containing 1 µM ionomycin and
30 mM LiOAc. Transfer the cells to 50ml of cold transport
buffer with 5% newborn calf serum. Centrifuge
for 5 min at 300g at 4°C and wash once in 50ml transport
- Resuspend cells in complete transport buffer at
107/ml. Add digitonin to 100 µg/ml (1 µl of a 10% stock
per 107 cells). Leave on ice for 3 min and check permeabilization
with trypan blue. Dilute the cells to 50ml
with transport buffer to release soluble transport
factors and collect by centrifugation as described
- Preincubation (optional): Resuspend cells in
transport buffer containing 30 mM LiOAc at 107/ml and
add MgATP (1 mM), creatine phosphate (5mM) and
creatine phosphokinase (20 U/ml). Incubate for 15 min
in a 30°C water bath. Wash cells with transport buffer.
This step results in the depletion of additional transport
factors such as CRM1, rendering them rate limiting in
the subsequent reaction. The reporter protein largely
remains in the nucleus under these conditions.
- Resuspend cells in transport buffer at 3 x 107/ml.
Assemble 40-µl transport reactions in FACS tubes:
300,000 permeabilized cells (10 µl), ATP-regenerating
system (1mM MgATP, 5mM creatine phosphate,
20U/ml creatine phosphokinase), Cy5-BSA-NLS
(25-50µg/ml), l bOVl annealed oligonucleotide, and
HeLa cytosol (2mg/ml).
- Incubate tubes in a 30°C water bath for 30min.
Control reactions contain the same components but are
kept on ice, as nuclear import and export are temperature
- Stop the reaction by adding 4 ml of cold transport
buffer. Centrifuge for 5 min at 400g and 4°C. Remove
most of supernatant by aspiration and resuspend cells
in residual buffer. Proceed to step 9 or fix cells for
microscopic analysis by the addition of 2ml of
formaldehyde (3.7% in PBS). Incubate cells for 15min
at room temperature, add 1 µl Hoechst 33258 (10
mg/ml in H2O), and incubate for 5 more minutes.
Collect cells by centrifugation (300g for 5 min), wash
twice with 1 ml PBS, and resuspend the final cell pellet
in 15 µl PBS. Apply the cell suspension to a glass slide,
cover with a coverslip, and seal with nail polish.
- Measure the fluorescence of 10,000 cells by flow
cytometry. In Becton-Dickinson instruments, GFPNFAT
(or BSA-NLS, labeled with FITC or Cy2, if only
import is analyzed) is detected in FL1 and Cy5-BSANLS
- Normalize the mean fluorescence values with
respect to a reaction kept on ice.
Several of the basic controls for assaying nuclear
import in digitonin-permeabilized cells are shown
(Fig. 2). Nuclear import in digitonin-permeabilized cells is stimulated by the addition of HeLa cytosol or
reticulocyte lysate, which provides a source of factors,
including import receptors and Ran. A low level of
cytosol-independent import is usually observed
because digitonin permeabilization does not result in
the quantitative release of transport factors. Nuclear
import is inhibited at low temperature or by incubation
with wheat germ agglutinin (0.2-0.5mg/ml), a
lectin that binds to NPC proteins and blocks translocation
through the pore. The level of nuclear import is
quantified by measuring the nuclear fluorescence in
50-100 cells per condition using a commercially available
program such as OpenLab or using a public
domain program such as ImageJ (http://rsb.info.
|FIGURE 2 Nuclear import of FITC-BSA-NLS analyzed by fluorescence
microscopy. (Top) NIH 3T3 cells were digitonin permeabilized
and incubated with the indicated components (BUF, buffer; RL,
reticulocyte lysate; WGA, wheat germ agglutinin). DAPI staining of
DNA and phase-contrast (PHASE) images are also shown. Note that
FITC-BSA-NLS binds nonspecifically to cells in the absence of
cytosol. The rim fluorescence in the presence of WGA reflects the
arrest of FITC-BSA-NLS in import complexes at the NPC. (Bottom)
The level of nuclear import observed under each condition was
quantified from 12-bit images. Values reflect the average fluorescence
intensity per pixel from at least 100 nuclei per condition. Data
courtesy of Leonard Shank (University of Virginia).
The same controls that apply to nuclear import are
also used to validate nuclear export reactions (Fig. 3).
Export of GFP-NFAT is stimulated by the addition of cytosol or reticulocyte lysate and is inhibited at low
temperature or by the addition of wheat germ agglutinin.
Note that the preexport level of GFP-NFAT fluorescence,
measured in cells that have been kept on ice,
will vary depending on the cellular expression level.
Therefore, a reliable quantification of nuclear export
requires analysis of a large number of cells in order to
obtain statistically meaningful results. To this end, we
use flow cytometry to measure the residual fluorescence
in 10,000 cells (Fig. 4). This approach allows
rapid analysis of a large number of samples, e.g., for
determination of the transport kinetics (Fig. 4a) or the
cytosol dependence of transport (Fig. 4b). Nuclear
import of a fluorescently labeled import substrate can be analyzed simultaneously, allowing a direct comparison
between different transport pathways.
Nuclear import and nuclear export can be reconstituted
using recombinant factors instead of cytosol, an
approach that allows the contributions of individual
transport factors to be analyzed (Görlich et al.
Black et al.
, 2001; Kehlenbach and Gerace, 2002).
|FIGURE 3 Nuclear export of GFP-NFAT was analyzed by fluorescence
microscopy. GFP-NFAT cells were treated with trichostatin
A and ionomycin to induce expression and nuclear import of GFP-NFAT.
After digitonin permeabilization, the cells were incubated
with the indicated components (BUF, buffer; RL, reticulocyte lysate;
CYTO, HeLa cytosol; WGA, wheat germ agglutinin). Hoechst staining
of DNA and phase-contrast (PHASE) images are also shown.
|FIGURE 4 Nuclear export of GFP-NFAT and nuclear import of
Cy5-BSA-NLS were analyzed in parallel by flow cytometry. (a) Time
course of nuclear transport. All reactions contained 2mg/ml of
cytosol and 25µg/ml of recombinant Ran. (b) Cytosol dependence
of nuclear transport. Reactions contained 50µg/ml of recombinant
Ran and the indicated amounts of cytosol. All reactions were performed
using preincubated cells (see step 5 in Section III,E) to
enhance the cytosol dependence of transport. Portions of this figure
were reprinted by permission from the J. Cell Biol., Rockefeller University
Any compromise in the integrity of the nuclear
envelope renders permeabilized cell assays uninterpretable.
This could occur if cells are overpermeabilized
with digitonin. Under such a condition,
NLS-containing reporters can appear to undergo
nuclear import when they are, in fact, simply binding
to DNA after leakage through a permeabilized nuclear
envelope. Likewise, NES-containing reporters can
appear to undergo export due to simple leakage from
the nucleus. The easiest way to establish that nuclear
import or export is mediated by the NPC is to test for
inhibition by WGA. Alternatively, the intactness of the
nuclear envelope can be demonstrated by showing
that a fluorescently labeled dextran (≥70kDa) is
excluded from the nucleus. Thus, it is helpful to test a
range of digitonin concentrations (25-100µg/ml) and
stain with trypan blue to optimize permeabilization of
the plasma membrane.
The assays described in this article feature NIH 3T3
and HeLa cells; however, these methods should be
applicable to virtually any mammalian cell line. Digitonin
permeabilization on adherent cells works best
when the cells are 40-70% confluent and poorly if the
cells are approaching confluence. Cells that are not
well adhered may detach during the permeabilization and wash steps, a problem that usually can be overcome
by coating coverslips with poly-D
-lysine or by
plating the cells 2 days before permeabilization. Also,
because cells near the edge of the coverslip may be
subject to evaporation artifacts even in a humid
chamber, it is best to restrict analysis to the central
region of the coverslip.
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