Imaging Techniques Cell Biology Biochemistry

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Authors: Hiromi Tissera, Mohamed Kodiha & Ursula Stochaj

Abstract

Many protocols designed to analyze the intracellular distribution of antigens depend on detergents that permeabilize or semi-permeabilize membranes (1). 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) intact (2). 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 vitro (3). 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 cells (3); 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.

Reagents

  1. Bovine serum albumin (BSA)
  2. 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)]
  3. Cell culture medium (Gibco)
  4. Digitonin (Sigma)
  5. DMSO (Sigma)
  6. Formaldehyde
  7. Mounting medium; for example Vectashield (Vector Laboratories)
  8. NaN3
  9. PBS, phosphate-buffered saline, pH 7.4
  10. Triton X-100
  11. Antibodies against a protein located in the nucleus; here: polyclonal anti-lamin B antibodies (Santa Cruz Biotechnology, sc-6217)
  12. Cy3-labeled affinity-purified secondary antibodies (Jackson ImmunoResearch) 4’,6-diamidino-2-phenylindole (DAPI; Sigma)

Cell lines

  1. HeLa S3 (human, cervix carcinoma)
  2. LLC-PK1 (pig, kidney)
  3. NIH3T3 (mouse, fibroblast)

Equipment

  1. CO2 incubator
  2. Laminar flow hood
  3. Humidity chamber
  4. Confocal microscope (for example: Zeiss LSM510) or fluorescence microscope equipped with appropriate camera.

Procedure

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.

  1. Dissolve digitonin in DMSO; prepare stock solutions in DMSO that are 1,000 fold concentrated. Immediately before use, dilute digitonin into buffer B.
  2. Incubate cells for 3 min at room temperature and remove liquid.
  3. Wash once with cold buffer B, rinse with PBS and fix with 3.7% formaldehyde in PBS for 25 min.
  4. 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.
  5. Block with PBS/2 mg/ml BSA/1 mM NaN3 (blocking solution) for 1 h.
  6. Incubate overnight with anti-lamin B antibodies (0.5 μg/ml) in blocking solution.
  7. 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.
  8. Wash 3 times for 10 min with blocking solution, then incubate with 1 μg/ml DAPI in blocking solution for 2 min.
  9. 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 averaging (9).

PROCESSING OF IMAGES

Process images with appropriate software such as Adobe Photoshop 8.0.

Timing

The procedures can be completed in 18 hours.

Troubleshooting

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 immunostaining (10).

References

  1. Jamur, M.C. & Oliver, C. Permeabilization of cell membranes. in Methods in Molecular Biology 588, 63-66 (2009).
  2. 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).
  3. 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)
  4. Weis, K. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441-451 (2003).
  5. Wagstaff, K.M. & Jans, D.A. Importins and beyond: non-conventional nuclear transport mechanisms. Traffic 10, 1188-1198 (2009).
  6. Kodiha, M., Crampton, N., Shrivastava, S., Umar R. & Stochaj, U. Traffic control at the nuclear pore. Nucleus 1, 237-244 (2010).
  7. 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.
  8. 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).
  9. 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).
  10. 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).

Acknowledgements

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

Figures

Figure 1: Effect of digitonin on the integrity of nuclear membranes in NIH3T3, LLC-PK1 and HeLaS3 cells.

Download Figure 1

NIH3T3, LLC-PK1 and HeLaS3 cells were treated with 5 or 40 μg/ml digitonin as indicated. Cells were fixed and subsequently processed for indirect immunofluorescence with antibodies against lamin B; DNA was stained with DAPI. In control experiments, cells were incubated with Triton X-100 before the blocking step. Note that with 40 μg/ml digitonin weak staining with lamin B antibodies is observed for NIH3T3 and LLC-PK1, but not for HeLa S3 cells. Arrowheads indicate the staining of lamin B in NIH3T3 and LLC-PK1 cells that were incubated with 40 μg/ml digitonin. Size bar is 20 μm.

Author information

Hiromi Tissera, Mohamed Kodiha & Ursula Stochaj, McGill University

Correspondence to: Ursula Stochaj ([email protected])

Source: Protocol Exchange (2010) doi:10.1038/protex.2010.211. Originally published online 23 December 2010.

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