By Mitotic Xenopus Egg Extract in Semi Intact MDCK Cells

Fumi Kano, Katsuya Takenaka, and Masayuki Murata

Summary

Semi-intact cells are cells with plasma membranes that have been permeabilized by bacterial pore-forming toxins or surfactants. The addition of mitotic Xenopus egg extract to semi-intact cells can reconstitute a number of intracellular events that occur specifically at the onset of mitosis. In this chapter, we describe methods for reconstituting the disassembly of the Golgi apparatus by introducing mitotic Xenopus egg extract into semi-intact Mardin-Darby canine kidney (MDCK) cells. The Golgi apparatus was visualized in the cells by expression of green fluorescence protein (GFP)-tagged galactosyltransferase, a marker of trans-Golgi cisternae. Xenopus egg extracts arrested at mitosis or interphase were then prepared and added to the semi-intact MDCK cells. Disassembly of the Golgi apparatus was induced by mitotic Xenopus egg extract. This system can be used not only to elucidate the factors that are involved in the reconstitution process, but also to dissect the process into several elementary steps morphologically and biochemically.

Key Words: GFP; Golgi apparatus; kinase; mitosis; reconstitution; semi-intact cell; Xenopus egg extract.

1. Introduction

Semi-intact cells are cells with plasma membranes that have been permeabilized by bacterial pore-forming toxins or detergents. Various intracellular events can be functionally reconstituted in semi-intact cells by incubation with cytosol and adenosine triphosphate (ATP; Fig. 1). Because semi-intact cells allow direct access of chemicals and antibodies to the cytoplasm of the cells, they permit study of the molecular mechanisms of the reconstituted process biochemically. We used a bacterial cytolysin, streptolysin O (SLO), to permeabilize the cells (1-3). SLO binds to cholesterol in plasma membranes at 4°C. At warmer temperatures, SLO assembles to form amphiphilic hexamers, resulting in the generation of small, stable transmembrane pores (4).

SLO-induced pores are 30 nm in diameter, which is sufficient to allow entry of immunoglobulin into the cells (immunoglobulin G; 150 kDa) (5). We can minimize

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

GFP-tagged proteins or organelles

Cytosol and ATP

To study the mechanisms of the reconstituted process, add

■ recombinant proteins (dominant active or negative form)

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o o O

O

+SLO

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Cytosol + ATP

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Visualization of intracellular events in intact cells

Permeabiiization by SLO

Reconstitution of intracellular events in semi-intact cells

Fig. 1. Scheme of semi-intact cells.

the damage to membranes of intracellular organelles caused by passage of SLO through the SLO-induced pores in the plasma membrane into the cytoplasm by washing away any excess SLO at 4°C before the initiation of pore formation. In contrast, it is difficult to avoid damage to intracellular structures when cells are permeabilized with digitonin because digitonin permeabilization is insensitive to temperature. SLO-mediated semi-intact cells have thus proven useful for the reconstitution of transport to apical or basolateral membranes in polarized cells (6,7), the import of proteins to peroxisomes (8), and so on.

This chapter presents protocols for the reconstitution of the disassembly of the Golgi apparatus by mitotic Xenopus egg extract in SLO-induced, semi-intact Mardin-Darby canine kidney (MDCK) cells.

2. Materials

1. Marc's modified Ringer's (MMR): 100 mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM Na-HEPES, pH 7.8, 0.1 mM ethylenediaminetetraacetic acid.

2. EGFP-N1 vector (Clontech; Palo Alto, CA).

3. LipofectAMINE PLUS (Invitrogen; Carlsbad, CA).

4. Geneticin (Gibco-BRL).

5. Transwell (24-mm diameter, 0.4-|lm pore size; Corning Costar).

6. Transport buffer (TB): 25 mM HEPES-KOH, pH 7.4, 115 mM potassium acetate, 2.5 mM MgCl2, 1 mM dithiothreitol, and 2 mM O,O 9-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA); store at 4°C.

7. ATP (Sigma): 100 mM stock in water; store at -30°C.

8. Creatine phosphate (Sigma): 800 mM stock in water; store at -30°C.

9. Creatine kinase (Sigma): 5-mg/mL stock in 50 %glycerol; store at -30°C.

10. PD98059 (New England Biolabs): 50-mg/mL stock in dimethyl sulfoxide (DMSO); store at -30°C.

11. Staurosporine (Wako): 25 mg/mL stock in DMSO; store at -30°C.

12. SB203580 (Calbiochem): 20 mg/mL stock in DMSO; store at -30°C.

13. Butyrolactone I (Affiniti Research Products): 100 mg/mL stock in DMSO; store at -30°C.

14. Protease inhibitors (Sigma): chymostatin and pepstatin A, 5-mg/mL stock in water; leupeptin and antipain, 5 mg/mL stock in DMSO; store at -30°C.

15. Paclitaxel (Taxol) (Sigma): final concentration 5 |lg/mL, prepared just before experiments.

16. SLO (purchased from Dr. Bhakdi, Meintz University, Germany).

17. Propidium iodide (Molecular Probes): 1.0-mg/mL stock in water; store at 4°C

18. Mouse anti-cdc2 kinase antibody (Santa Cruz Biotechnology).

19. Protein G and protein A-Sepharose (Amersham Pharmacia).

20. 33°C incubator.

21. Zeiss LSM510 confocal microscope (Carl Zeiss Inc.).

22. OptimaTM TLX (Beckman Instruments Inc.).

3. Methods

3.1. Preparation of Xenopus Egg Extracts

Xenopus egg extracts were prepared as previously described except that cytochala-sin B was omitted (9,10).

1. M phase (cytostatic factor arrested) extracts are prepared from unfertilized eggs, and interphase extracts are prepared from parthenogenetically activated eggs that are electrically stimulated in 0.2X MMR by two 1-s pulses of 12 V alternating current followed by incubation for 10 min at room temperature. Eggs can also be activated with 0.1 |lg/mL A23187 in 0.5X MMR for 3 min.

2. Dilute the prepared extracts sevenfold with transport buffer containing protease inhibitors.

3. Centrifuge the extracts in the Optima TLX at 100,000g for 60 min at 4°C.

4. The supernatant is collected and stored at -80°C (see Note 1).

3.2. Visualization of the Golgi Apparatus in MDCK Cells

The Golgi apparatus in mammalian cells consists of stacked cisternae and tubular networks in the perinuclear region during interphase but diffuses throughout the cytoplasm at the onset of mitosis (11,12). In polarized MDCK cells, the Golgi apparatus appears as a ribbonlike structure adjacent to the nucleus during interphase. To visualize the Golgi apparatus in MDCK cells, we stably express galactosyltransferase (GT), a marker for trans-Golgi cisternae and the trans-Golgi network, fused to GFP (green fluorescence protein; GT-GFP).

3.2.1. Cloning

1. Complementary DNA (cDNA) encoding the first 60 amino acids of mouse GT is amplified from a mouse liver cDNA library using the polymerase chain reaction (PCR).

2. Insert the PCR fragment in-frame upstream of the EGFP cDNA in the EGFP-N1 vector.

3. MDCK cells are transfected by LipofectAMINE PLUS according to the manufacturer's instructions.

3.2.2. Selection of MDCK Cell Lines Stably Expressing GT-GFP

1. Select stable transfectants (MDCK-GT) in complete medium containing 300 | g/mL Geneticin.

2. After 10 d, surviving cell colonies are isolated and screened visually for Golgi-localized fluorescence. Several positive clones are identified and expanded into cell lines for further experiments.

3.3. Reconstitution of Golgi Disassembly in Semi-Intact MDCK Cells

3.3.1. Preparation of Semi-Intact Cells (see Note 2)

1. Add 1 x 106 MDCK-GT cells to the upper chamber of each Transwell. Add 2.5 mL culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum) to the lower chamber. The cells are incubated in a 37°C/5% CO2 incubator for 3 to 4 d to form a tight polarized monolayer.

2. Incubate the cells with 5 |lg/mL Taxol for 30 min at 37°C to stabilize microtubules.

3. Transfer the upper chamber to a 6-cm dish. Add 0.5 mL of 500 ng/mL preactivated SLO to the upper chamber for 20 min at 4°C.

4. After washing out the excess SLO three times with ice-cold phosphate-buffered saline, add 1 mL prewarmed TB containing 3 |g/mL propidium iodide for 20 min at 37°C to form pores in the plasma membranes. Of the total cytosol, 60% leaks out under these conditions, as determined by measurement of the leakage of cytosolic lactate dehydrogenase (13; see Note 3). SLO-induced pores can be resealed with Ca2+ (see Note 4).

3.3.2. The Golgi Disassembly Assay

1. Incubate the permeabilized MDCK-GT cells with 1 M KCl in TB for 5 min at 4°C to remove peripheral membrane proteins.

2. Remove the polycarbonate membrane from the plastic side wall of the upper chamber with a knife and immerse it in TB with the cells facing upward. Cut the membranes into fragments 10 x 6 mm2 and cut the lower right-hand corner of the pieces of the membranes to identify the surface on which the cells are cultured.

3. Incubate the fragments with 20 |L reaction mixture at 33°C for 80 min (see Note 5). The reaction mixture contains mitotic Xenopus egg extract (diluted to 4.5 to 5.5 mg proteins/ milliliter in TB), ATP-regenerating system (1 mM ATP, 8 mM creatine kinase, and 50 |g/mL creatine phosphate), 1 mM guanosine 5'-triphosphate (GTP), and 1 mg/mL glucose.

4. Fix cells with 1% formaldehyde in TB for 20 min at room temperature.

5. View cells with an LSM 510 confocal microscope system.

3.4. Morphometric Analysis

1. The disassembly of the Golgi apparatus can be classified into three stages based on the fluorescence microscopic observations and the morphological properties of the Golgi membranes at each stage are easily distinguished in the same manner (Fig. 2):

a. Stage I (intact): a ribbonlike structure of the Golgi apparatus at the apical side of the cell.

b. Stage II (punctate): fragmented, punctate Golgi membranes. By electron microscopic observation, the punctate structures have an 0.8-|m average length and associate with microtubules at the apical side of the cells.

c. Stage III (dispersed): completely dispersed profiles of Golgi membranes. The Golgi membranes disperse throughout the cytoplasm. Some of the GT-GFP relocates to the endoplasmic reticulum (ER) membranes, which are identified by colocalization of GT-GFP with protein disulfide isomerase, a specific ER marker.

2. Count the number of cells at each stage. Be careful not to include nonpermeabilized cells, which are not stained by propidium iodide. We usually perform three independent experiments and calculate the means and standard deviations of the number of cells at stages I to III. Morphological changes of Golgi membranes can be expressed as the percentage of cells at stages I to III.

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