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Reconstitution of Golgi Disassembly 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 cistemae. 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 4C. 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 kOa) (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

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Kano, Takenaka, and Murata

To study the mechanisms of the reconstituted process, add ' chemicals (e.g. Inhibitors) antibody ' recombinant proteins (dominant active or negative form)

GFP-tagged proteins or organelles

Cytosol and ATP

+SLO
Visualization of intracellular events in intact cells

Cytosol + ATP Reconstitution of intracellular events in semi-intact cells

Permeabilization bySLO

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 4C 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. SLOmediated 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): lOOmMNaCl, 2 mMKCl, 1 mMMgS04, 2 mMCaClj, 5 mMNa-HEPES, pH 7.8, 0.1 mA/ethylenediaminetetraacetic acid. 2. EGFP-Nl vector (Clontech; Palo Alto, CA). 3. LipofectAMINE PLUS (Invitrogen; Carlsbad, CA). 4. Geneticin (Gibco-BRL). 5. Transwell (24-mm diameter, 0.4-nm pore size; Corning Costar). 6. Transport buffer (TB): 25 mM HEPES-KOH, pH 7.4, 115 mM potassium acetate, 2.5 mM MgCL, 1 mM dithiothreitol, and 2 mM 0,0 9-Z?w(2-aminoethyl)ethyleneglycolAf.MA^'.Af'-tetraacetic acid (EGTA); store at 4C. 7. ATP (Sigma): 100 mM stock in water; store at -30C. 8. Creatine phosphate (Sigma): 800 mM stock in water; store at -30C. 9. Creatine kinase (Sigma): 5-mg/mL stock in 50 %glycerol; store at -30C. 10. PD98059 (New England Biolabs): 50-mg/mL stock in dimethyl sulfoxide (DMSO); store at -30C. 11. Staurosporine (Wako): 25 mg/mL stock in DMSO; store at -30C. 12. SB203580 (Calbiochem): 20 mg/mL stock in DMSO; store at -30''C.

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13. Butyrolactone I (Affiniti Research Products): 100 mg/mL stock in DMSO; store at -30C. 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 -30C. 15. Paclitaxel (Taxol) (Sigma): final concentration 5 |j,g/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 4C 18. Mouse anti-cdc2 kinase antibody (Santa Cruz Biotechnology). 19. Protein G and protein A-Sepharose (Amersham Pharmacia). 20. 33C incubator. 21. Zeiss LSM510 confocal microscope (Carl Zeiss Inc.). 22. OptimaTM TLX (Beckman Instruments Inc.).

3. Methods 3.1. Preparation ofXenopus Egg Extracts

Xenopus egg extracts were prepared as previously described except that cytochalasin 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 |a,g/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 100,0G0g for 60 min at 4C. 4. The supernatant is collected and stored at -80C (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-Nl 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 |Xg/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.

360 3.3. Reconstitution 3.3.1. Preparation

Kano, Takenaka, and Murata of Golgi Disassembly in Semi-Intact of Semi-Intact Cells (see Note 2) MDCK Cells

1. Add 1 X lO*" 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 37C/5% CO, incubator for 3 to 4 d to form a tight polarized monolayer. 2. Incubate the cells with 5 Hg/mL Taxol for 30 min at 37C 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 4C. 4. After washing out the excess SLO three times with ice-cold phosphate-buffered saline, add 1 mL prewu-med TB containing 3 |Xg/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 Ca^"^ {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 4C 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 mm^ 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 |J,L reaction mixture at 33C 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 |ig/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-nm 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.

stage I (intact)

stage II (punctate)

stage III (dispersed)

Fig. 2. Morphological dissection of the Golgi disassembly in semi-intact cells. Cells at stages I (intact), II (punctate), and III (dispersed) were observed by confocal (A-C) and electron (D-F) microscopy. In A-C, the lower panels show x-y images at the apical side of the cell, and the upper panels show x-z sectioning images. (A,D) Stage I (intact). Semi-intact MDCK-GT cells were incubated with TB and ATP at 33C for 80 min. A large stack of cisternae of the Golgi apparatus is seen at the apical side of the cell. (B,E) Stage II (punctate) after 80-min incubation with mitotic Xenopus egg extracts containing cdc2 kinase inhibitor (stage II). Ministacked Golgis (MSG) are seen at the apical side of the cell, where tight junction (TJ) is observed. Ministacked Golgi associates with microtubules (MT). (F) Stage III (dispersed) 80 min after incubation with mitotic Xenopus egg extracts. No stacked structure of the Golgi apparatus is observed, and only tubulovesicular structures (arrows), which probably derived from the Golgi, are seen. Inset represents the tubulovesicular structures (asterisk) at a higher magnification. Bars: (A-C) 20 nm; (D-F) 1 |j,m. Reproduced with permission from ref. 2. 2000 The Rockefeller University Press.

362 100 80 o *->

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Kano, Takenaka, and Murata

intact H punctate D dispersed x

60

o 40 o
20 0 cont SS BL PD SB BL+PD

Fig. 3. Effect of protein kinase inhibitors on the Golgi disassembly. Semi-intact MDCK-GT cells were incubated with mitotic Xenopus egg extracts and ATP containing no inhibitor (control, cont), staurosporine (SS), butyrolactone 1 (BL), PD98059 (PD), SB203580 (SB), or BL + PD, respectively, at 33C for 80 min. The cells were fixed, and morphometric analysis was performed. Reproduced with permission from ref. 2. 2000 The Roclcefeller University Press.

3.5. Biochemical Analysis for the Golgi Disassembly 3.5.1. Kinase Inhibitors
1. Incubate 1 M KCl-washed, semi-intact MDCK-GT cells with mitotic Xenopus extract, ATP-regenerating system, GTP, and glucose in the presence of protein inhibitors, 25 \i.M staurosporine (protein kinase inhibitor), 20 \xM SB203580 (p38 mitogen-activated protein kinase inhibitor), 50 [iM PD98059 (mitogen-activated protein kinase kinase [MEK] inhibitor), 30 \JiM butyrolactone 1 (cdc2 kinase inhibitor) at 33C for 80 min. 2. Fix the cells with 1 % formaldehyde and view by confocal microscopy. 3. Count the number of cells at stages 1 to III and calculate the percentage of cells at each stage (Fig. 3).

3.5.2. Immunodepletion

of Kinases From Mitotic Xenopus Extracts

1. Incubate 20 |J,L mouse anti-cdc2 antibody and 30 |iL protein G-Sepharose or 30 |a.L of rabbit anti-MEK serum and 30 |xL of protein A-Sepharose at 4C for 1 h with rotation. 2. Wash antibody-coupled beads with TB containing protease inhibitors (25 (ig/mL chymostatin, pepstatin A, leupeptin, and antipain) three times. 3. Incubate 150 jiL of mitotic Xenopus egg (4.5-5.5 mg proteins/mL) extracts with antibody-coupled beads at 4C for 1 h with rotation. 4. Remove the beads by centrifugation (2300^ or 5000 rpm, 4C, 30 s) and transfer the supernatant to a new tube. 5. Add fresh antibody-coupled beads to the supernatant and incubate at 4C for 1 h with rotation. 6. Repeat steps 4 and 5 twice in preparation of cdc2-depleted mitotic Xenopus extracts. 7. Remove the beads. Store the supernatant (cdc2- or MEK-depleted mitotic Xenopus extracts) at -80C. 8. Check the depletion of cdc2 or MEK from mitotic Xenopus extracts by Western blotting. 9. Incubate IM KCl-washed, semi-intact MDCK-GT cells with cdc2- or MEK-depleted Xenopus extract, ATP-regenerating system, GTP, and glucose at 33C for 80 min.

Reconstitution

of Mitotic

Golgi 100-

Disassembly intact s punctate dispersed

363

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-in

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Fig. 4. Inhibition of the Golgi disassembly by cdc2- or MEK-depleted Xenopus egg extracts. Mock, cdc2- or MEK-depleted Xenopus egg extracts were applied to semi-intact cells and incubated at 33C for 80 min. The cells were fixed, and morphometric analysis was performed. Cdc2-depleted extracts arrested the disassembly process at stage II (punctate), and MEKdepleted extracts did so at stage I (intact). Reproduced with permission from ref. 2. 2000 The Rockefeller University Press.

Stage I (intact)

Stage II (punctate)

Stage III (dispersed)

Fig. 5. Schematic model of Golgi disassembly.

10. Fix the cells with 1% formaldehyde and view by confocal microscopy. 11. Count the number of cells at stages I to III and calculate the percentage of cells at each stage (Fig. 4). From the results using the Golgi disassembly assay based on the semi-intact cell system, Golgi disassembly is dissected into two sequential elementary steps, morphologically and biochemically (Fig. 5). The Golgi apparatus appears as a ribbonlike structure at the apical side of the cell but then fragments to form punctate structures in a MEK-dependent manner (stage I to II), followed by dispersion of the Golgi membranes and relocation to the ER in a cdc2-dependent manner (stage II to III).

364 4. Notes

Kano, Takenaka, and Murata

1. MPF (M phase-promoting factor) activity varies between extracts. It can be measured by assaying phosphorylation of histone HI (9,10). 2. The permeability of the cells can be altered by various experimental conditions: cell density, temperature, incubation time, cell type, and so on. It is important to maximize permeabilization before starting the experiments. 3. We use propidium iodide to check the permeability of the cells. Propidium iodide is a cell-impermeable dye that stains nucleic acids and can be excited with either mercury or xenon arc lamps or with an argon ion laser (Ex/Em = 535/617 [nm/nm]), making it suitable for two-color imaging in combination with GFP or fluorescein derivatives. Other impermeable dyes or fluorescence-labeled proteins, such as trypan blue, eosin, azure A, or tetramethylrhodamine isothiocyanate isomer R (TRITC)-labeled phalloidin, can be used to check the permeability of the cells (1,14.15). Leakage of endogeneous cytosol can be examined by monitoring the activity of lactate dehydrogenase (135 kDa), a cytoplasmic enzyme, as described in refs. 8 and 13. 4. SLO-induced pores can be resealed with Ca^+ as demonstrated in refs. 16 and 17. By resealing pores after exposure of semi-intact cells with nuclear extracts and cytosol prepared from different cell types, the properties and fates of cells could be altered to other ones (17,18). 5. Because the assay is performed in 20 |xL reaction mixture, we need to remove the TB from the membranes completely before incubation with the reaction mixture. We usually place a fragment of the membrane on a Kimwipe for a short time to absorb the TB and then transfer it on parafilm before quickly applying the reaction mixture. To avoid drying during the incubation at 33C, it is necessary to cover the fragments with the lid of the culture dish. In addition, we place small pieces of wet Kimwipe under the cover to minimize moisture loss. References 1. Kano, P., Takenaka, K., Yamamoto, A., Nagayama, K., Nishida, E., and Murata, M. (2000) MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopiis extracts. J. Cell Biol. 149, 357-368. 2. Kano, P., Sako, Y., Tagaya, M., Yanagida, T., and Murata, M. (2000) Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semiintact Chinese hamster ovary cells. Mol. Biol. Cell. 11, 3073-3087. 3. Kano, P., Nagayama, K., and Murata, M. (2000) Reconstitution of the Golgi reassembly process in semi-intact MDCK cells. Biophys. Chem. 84, 261-268. 4. Bhakdi, S., Tranum-Jensen, J., and Sziegoleit, A. (1985) Mechanism of membrane damage by streptolysin-O. Infect. Immunol. 47, 52-60. 5. Bhakdi, S., Weller, U., Walev, I., Martin, E., Jonas, D., and Palmer, M. (1993) A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med. Microbiol. Immunol. (Bed.) 182, 167-175. 6. Lafont, P., Burkhardt, J. K., and Simons, K. (1994) Involvement of microtubule motors in basolateral and apical transport in kidney cells. Nature 372, 801-803. 7. Pimplikar, S. W., Ikonen, E., and Simons, K. (1994) Basolateral protein transport in streptolysin O-permeabilized MDCK cells. J. Cell Biol. 125, 1025-1035. 8. Wendland, M. and Subramani, S, (1993) Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J. Cell Biol. 120, 675-685.

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