Nuclear envelopes show cell-type specific sensitivity for the permeabilization with digitonin.

doi:10.1038/protex.2010.211

Method Article

Nuclear envelopes show cell-type specific sensitivity for the permeabilization with digitonin.

https://doi.org/10.1038/protex.2010.211

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Many protocols designed to analyze the intracellular distribution of antigens depend on detergents that permeabilize or semi-permeabilize membranes¹ . Whereas Triton X-100 or similar detergents are used to permeabilize all cellular membranes, digitonin can permeabilize preferentially the plasma membrane of mammalian culture cells under conditions that leave the nuclear envelope (NE) intact² . Digitonin treatment of fixed cells has been important to distinguish between antigens on the nuclear and cytoplasmic side of the NE. Furthermore, incubation of unfixed cells with digitonin is a critical step to analyze nuclear import in vitro³ . Macromolecules move in and out of the nucleus through nuclear pore complexes (NPCs) which are embedded in the NE^4-6. For the analysis of nuclear transport in a cell-free system it is therefore mandatory that the NE remains intact during the incubation period. Many in vitro nuclear transport studies were carried out with HeLa cells³; however, for some applications the use of other cell lines is preferable. Here, we compare the permeabilization of NEs in three mammalian cell lines that were treated with digitonin. Our protocol provides a guide to optimize the concentration of digitonin to permeabilize the plasma membrane, but not the NE, of mammalian culture cells.

Biochemistry

Cell biology

Biological techniques

Computational biology and bioinformatics

immunofluorescence

nuclear envelope

digitonin

membrane permeabilization

Bovine serum albumin (BSA)

Buffer B, adjusted to pH 7.3 with NaOH [20mM Hepes; 110 mM potassium acetate; 5 mM sodium acetate; 2 mM magnesium acetate; 1 mM EGTA; add immediately before use: 2 mM DTT and protease inhibitors (aprotinin, leupeptin, pepstatin; each at 1μg/ml)]

Cell culture medium (Gibco)

Digitonin (Sigma)

DMSO (Sigma)

Formaldehyde

Mounting medium; for example Vectashield (Vector Laboratories)

NaN₃

PBS, phosphate-buffered saline, pH 7.4

Triton X-100

Antibodies against a protein located in the nucleus; here: polyclonal anti-lamin B antibodies (Santa Cruz Biotechnology, sc-6217)

Cy3-labeled affinity-purified secondary antibodies (Jackson ImmunoResearch)

4',6-diamidino-2-phenylindole (DAPI; Sigma)

Cell lines

HeLa S3 (human, cervix carcinoma)

LLC-PK1 (pig, kidney)

NIH3T3 (mouse, fibroblast)

CO₂ incubator

Laminar flow hood

Humidity chamber

Confocal microscope (for example: Zeiss LSM510) or fluorescence microscope equipped with appropriate camera.

CELL CULTURE

Culturing of cells on poly-lysine coated coverslips has been described previously^7,8.

INDIRECT IMMUNOFLUORESCENCE

All incubations are carried out in a humidity chamber at room temperature.

Dissolve digitonin in DMSO; prepare stock solutions in DMSO that are 1,000 fold concentrated. Immediately before use, dilute digitonin into buffer B.
Incubate cells for 3 min at room temperature and remove liquid.
Wash once with cold buffer B, rinse with PBS and fix with 3.7% formaldehyde in PBS for 25 min.
Rinse with PBS; then move to step 5 or permeabilize with 0.1% Triton X-100 in PBS/2 mg/ml BSA/1 mM NaN₃ for 5 min.
Block with PBS/2 mg/ml BSA/1 mM NaN₃ (blocking solution) for 1 h.
Incubate overnight with anti-lamin B antibodies (0.5 μg/ml) in blocking solution.
Wash 3 times for 10 min with blocking solution, then incubate for 2 h with Cy3-labeled secondary antibodies (3 μg/ml) in blocking solution.
Wash 3 times for 10 min with blocking solution, then incubate with 1 μg/ml DAPI in blocking solution for 2 min.
Mount specimen and seal with nail polish.

IMAGE ACQUISITION

Acquire images by confocal microscopy (for example, Zeiss LSM510) using a 63x objective (oil, NA = 1.4), pixel resolution of 0.65 μm, scan speed 5, four-line averaging⁹.

PROCESSING OF IMAGES

Process images with appropriate software such as Adobe Photoshop 8.0.

The procedures can be completed in 18 hours.

Nuclear envelope is permeabilized by digitonin treatment

The concentration of digitonin as well as the incubation time with the detergent has to be optimized. For some applications it may be suitable to treat with digitonin on ice.

Intactness of NE cannot be tested by antibody staining

If it is not possible to test the barrier function of NEs by staining with antibodies against nuclear proteins, use alternative methods. For instance, the entry into the nucleus of large non-nuclear proteins, such as GFP-β-galactosidase, provides a method to detect leakiness of NEs without the need for immunostaining¹⁰.

Jamur, M.C. & Oliver, C. Permeabilization of cell membranes. in Methods in Molecular Biology 588, 63-66 (2009).
Griffis, E.R., Xu, S. & Powers, M.A. Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes. Mol. Biol. Cell 14, 600-610 (2003).
Adam, S.A., Marr, R.S. & Gerace, L. Nuclear protein import in permeabilized mammalian cells requires cytoplasmic factors. J. Cell Biol. 111, 807-816 (1990)
Weis, K. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441-451 (2003).
Wagstaff, K.M. & Jans, D.A. Importins and beyond: non-conventional nuclear transport mechanisms. Traffic 10, 1188-1198 (2009).
Kodiha, M., Crampton, N., Shrivastava, S., Umar R. & Stochaj, U. Traffic control at the nuclear pore. Nucleus 1, 237-244 (2010).
Kodiha, M., Tran, D., Morogan, A., Qian, C. & Stochaj, U. Dissecting the signaling events that impact classical nuclear import and target nuclear transport factors. PLoS One 4, e8420.
Kodiha, M., Tran, D., Qian, C., Morogan, A., Presley, J.F., Brown, C.M. & Stochaj, U. Oxidative stress mislocalizes and retains transport factor importin-alpha and nucleoporins Nup153 and Nup88 in nuclei where they generate high molecular mass complexes. Biochim. Biophys. Acta 1783, 405-418 (2008).
Kodiha, M., Brown, C.M. & Stochaj, U. Analysis of signaling events by combining high throughput screening technology with computer based image analysis. Sci. Signal. 1, p12 (2008).
Sanchez, L., Kodiha, M. & Stochaj, U. Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase. J. Biomol. Techn. 16, 235-238 (2005).

This work was supported by grants from CIHR, NSERC and HSFQ to US. MK is a supported by a postdoctoral fellowship from McGill University.

The authors declare no competing financial interests.

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Nuclear envelopes show cell-type specific sensitivity for the permeabilization with digitonin.

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Nuclear envelopes show cell-type specific sensitivity for the permeabilization with digitonin.

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Abstract

Figures

Reagents

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Procedure

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Acknowledgements

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