25 Reconstitution of Golgi Disassembly by Mitotic Xenopus Egg Extract in Semi-Intact MDCK Cells

8/13/2019 25 Reconstitution of Golgi Disassembly by Mitotic Xenopus Egg Extract in Semi-Intact MDCK Cells

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2 5Reconstitution of Golgi Disassemblyby M itotic XenopusEgg Extract in Semi-Intact MDCK CellsFumi Kano^ Katsuya Takenaka, and Masayuki Murata

SummarySemi-intact cells are cells with plasma membranes that have been permeabilized bybacterial pore-forming toxins or surfactants. The addition of mitotic Xenopusegg extractto semi-intact cells can reconstitute a number of intrace llular events that occur specificallyat the onset of mitosis. In this chapter, we describe methods for reconstituting the disassembly oftheGolgi apparatus by introducing m itoticXenopusegg extract into semi-intactMardin-Darby canine kidney (MDCK) cells. The Golgi apparatus was visualized in thecells by expression of green fluorescence protein (GFP)-tagged galactosyltransferase, a

marker of trans-Golgi cistemae. Xenopus egg extracts arrested at mitosis or interphasewere then prepared and added to the semi-intact MDCK cells. Disassembly of the Golgiapparatus was induced by mitoticXenopusegg extract. This system can be used not only toelucidate the factors that are involved in the reconstitution process, but also to dissect theprocess into several elementary steps morpho logically and biochem ically.Key Words: GFP; Golgi apparatus; kinase; mitosis; reconstitution; semi-intact cell;Xenopusegg extract.

1 . In t roduc t ionSemi-intact cells are cells with plasma m embrane s that have been perm eabilized bybacterial pore-forming toxins or detergents. Various intracellular events can be functionally reconstituted in semi-intact cells by incubation with cytosol and adenosinetriphosphate (ATP;Fig.1). Because semi-intact cells allow direct access of chemicalsand antibodies to the cytoplasm of the cells, they perm it study of the molecu lar m echa nisms ofthereconstituted process biochem ically. We used a bacterial cytolysin, streptolysin O (SL O), to permeabilize the cells (1-3). SLO binds to cholesterol in plasmamembranes at 4C. At warmer temperatures, SLO assembles to form amphiphilichexam ers, resulting in the generation of small, stable transmem brane pores (4).SLO-induced pores are 30 nm in diameter, which is sufficient to allow entry ofimmunoglobulin into the cells (immunoglobulin G; 150 kOa) (5). We can minimize

From:Method s in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal TransductionEdited by: X. J. Liu Hum ana Press Inc., T otow a, NJ357


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358 Kano, Takenaka, and Murata

GFP-tagged proteinsor organelles Cytosol and ATP

SLO

To study the m echanisms ofthe reconstituted process,addchemicals (e.g. Inhibitors) antibodyrecombinant proteins(dominant ac tive ornegative form)

CytosolATPVisualizat ion ofintracellular eventsin intact cells

Permeabilizationby SLO Rec onstitut ion ofintracellular eventsin semi-intact cellsFig. 1. Scheme of semi-intact cells.

the damage to membranes of intracellular organelles caused by passage of SLOthrough the SLO -induced pores in the plasma m embran e into the cytoplasm by washing away any excess SLO at 4C before the initiation of pore formation. In contrast, itis difficult to avoid damage to intracellular structures when cells are permeabilizedwith digitonin be cause digitonin permeabilization is insensitive to temperature. SLO -mediated semi-intact cells have thus proven useful for the reconstitution of transportto apical or basolateral membranes in polarized cells (6,7), the import of proteins toperoxisomes (8), and so on.

This chapter presen ts protocols for the reconstitution ofthedisassembly oftheGolgiapparatus by m itoticXenopus egg extract in SLO -induced, semi-intact Mardin-Darbycanine kidney (MDCK) cells.2 . Mater ials

1. Marc's modified Ringer's(MMR):lOOmMNaCl,2mMKCl,1mMMgS04,2mM CaClj,5 mMNa-H EPES, pH 7.8, 0.1 m A/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-nmpore size; Corning Costar).6. Transport buffer (TB ): 25 mM HEPES-KOH, pH 7.4, 115mM potassium acetate,2.5 mM MgC L,1 mM dithiothreitol, and2m M 0 , 0 9-Z?w(2-aminoethyl)ethyleneglycol-Af.MA^'.Af'-tetraacetic acid (EG TA); store at 4C.7. ATP (Sigma): 100mM stock in water; store at -3 0C.8. Creatine phosphate (Sigma): 800m M stock in w ater; store at -30 C .9. Creatine kinase (Sigma):5-mg/mLstock in 50 %glycerol; store at -30 C .10. PD98059 (New England Biolabs): 50-mg/mL stock in dimethyl sulfoxide (DMSO); storeat -30C .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 .


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Reconstitution of Mitotic Co lgi Disassemb ly 35913 . Butyrolactone I (Affiniti Research Produ cts): 100 mg /mL stock in DM SO ; store at -3 0 C .14 . Protease inhibitors (Sigm a): chym ostatin and pepstatin A, 5-mg /mL stoc k in wate r: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 expe rime nts.16. SLO (purchased from Dr. Bhakd i, M eintz University, Germ any).17. Propidium iodide (Mo lecular Probe s):1.0-mg/mL stock in water; store at 4C18. Mouse anti-cdc2 kinase antibody (Santa Cruz Biotechno logy).19 . Protein G and protein A-Seph arose (Amersham P harm acia).20. 33C incubator.21. Zeiss LSM 510 confocal microscope (Carl Zeiss Inc.).22. OptimaTM TLX (Beckman Instruments Inc.).

3. Methods3.1. Preparation ofXenopus gg xtracts

Xenopus egg ex t rac t s were p rep ared as p re v iou s ly desc r ibed excep t tha t cy to cha la-s in B was omi t t ed (9,10).

1. M phase (cytostatic factor arrested) extracts are prepa red from u nfertilized e ggs , 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 byincubation for 10 min at room temperature. Eggs can also be activated with 0.1 |a,g/mLA23187 in 0.5X MMR for 3 min.2. Dilute the prepared extracts sevenfold with transport buffer co ntaining protease inhibitors.3. Centrifuge the extracts in the Op tima TL X at 100,0G0g for 60 min at 4 C.4. The supernatant is collected and stored at -8 0 C (seeNote 1).

3.2. Visualization of the Golgi Apparatus in MDCK CellsThe Golg i appara tus in mammal ian ce l l s cons i s t s o f s t acked c i s t e rnae and tubu lar

ne tworks in the per inuc lear reg ion dur ing in te rphase bu t d i f fuses th roughout the cy to p lasm a t the onse t o f mi tos i s 11,12). In p o l a r i zed M D C K ce l l s , the Go lg i appa ra tusapp ears as a r ibbo n l ike s t ruc tu re ad jacen t to the nuc leus dur in g in te rph ase . To v i sua l i ze the Golg i appara tus in MDCK ce l l s , we s t ab ly express ga lac tosy l t rans ferase (GT) ,a marker fo r t rans -Golg i c i s t e rnae and the t rans -Golg i ne twork , fused to GFP (g reenf l u o re s cen ce p ro t e i n ; G T -G FP) .3.2.1. Cloning

1. Com plementary DN A (cDNA ) encoding the first 60 am ino acids of mo use 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 EG FP cDNA in the EG FP -N l vector.3. MD CK cells are transfected by LipofectAM INE PLU S according to the m anufa cturer's

instructions.3 2 2 Selection of MDCK Cell Lines Stably Expressing GT-GFP

1. Select stable transfectants (M DC K-G T) in com plete mediu m conta ining 300 |Xg/mLGeneticin.

2. After 10 d, surviving cell colonies are isolated and screen ed visually for G olgi-loca lizedfluorescence. Several positive clones are identified and expanded into cell lines for further experiments.


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360 Kano, Takenaka, and Murata3.3. Recons titution of Golgi Disassem bly in Sem i-Intact MD CK Cells3.3.1. Preparation of Sem i-Intact Cells see Note 2

1. Add 1 XlO* 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 calfserum) to the lower cham ber. Th e cells are incubated in a 37C /5% C O , incubator for 3 to4 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 cham ber to a 6-cm dish. Add 0.5 mL of 500 ng/mL preactivated SL Oto the upper chamber for 20 min at 4C.4. After wa shing out the excess SLO three times with ice-cold phosphate-buffered saline,add 1 mL prewu-med TB con taining 3 |Xg/mL propidiu m iodide for 20 m in at 37 C toform pores in the plasma membranes. Of the total cytosol, 60% leaks out under theseconditions, as determined by measurement of the leakage of cytosolic lactate dehydrogenase {13; see Note 3). SLO-induced pores can be resealed with Ca^ ^{see Note4).

3 3 2 The Golgi Disassembly Assay1. Incubate the permeabil ized M DC K-G T cells with 1M KCl in TB for 5 min at 4C toremove peripheral membrane proteins.2. Rem ove the polycarbon ate mem brane from the plastic side wall of the upper chamberwith a knife and immerse it in TB with the cells facing upward. Cut the membranes intofragments 10 x 6 mm^ and cut the lower right-hand corner of the pieces of the m em branes

to identify the surface on which the cells are cultured.3. Incub ate the fragmen ts w ith 20 |J,L reaction m ixture at 33C for 80 min {seeNote5). Thereaction mixture contains mitotic Xenopus egg extract (diluted to 4.5 to 5.5 mg proteins/milliliter in TB ), ATP -regenerating system(1mM A TP, 8 mM creatine kinase, and 50 |ig/mLcreatine phosphate), 1 mM gua nosine 5'-triphosphate (GTP), and 1 mg /mL glucose.4. Fix cells with 1% formaldehyd e in TB for 20 min at room tem perature .5. View cells with an LSM 5 10 confocal microscop e system.

3.4. Morphom etric Analysis1. The disasse m bly of the Golgi app aratus can be classified into three stages based on the

fluorescence microscopic observations and the morphological properties of the Golgimembranes 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 ofthecell.b. Stage II (punctate): fragmented, pun ctate Golgi me mb ranes. By electron microscopicobservation, the punctate structures have an 0.8-nm average length and associate withmicrotubules at the apical side of the cells.c. Stage III (dispersed): completely d ispersed profiles of Golgi me mb ranes. The Golgimembranes disperse throughout the cytoplasm. Some of the GT-GFP relocates to theendoplasmic 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 nonperm eabilizedcells, which are not stained by propidium iodide. We usually perform three independent experiments and calculate the means and standard deviations of the number ofcells at stages I to III. Morphological changes of Golgi membranes can be expressedas the percentage of cells at stages I to III.


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stage I (intact)

stage II (punctate)

stage III (dispersed)

Fig. 2. M orphological dissection of the Golgi disassem bly in sem i-intact cells. Cells at stagesI (intact), II (punctate), andIII(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 theupper panels show x-z sectioning images. (A,D) Stage I (intact). Semi-intact MDCK-GT cellswere incubated with TB and ATP at 33C for 80 min. A large stack of cisternae of the Golgiapparatus is seen at the apical side of the cell. (B,E) Stage II (punctate) after 80-min incubationwith mitotic Xenopus egg extracts containing cdc2 kinase inhibitor (stage II). MinistackedGolgis (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 afterincubation with mitotic Xenopus egg extracts. No stacked structure of the Golgi apparatus isobserved, and only tubulovesicular structures (arrow s), 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. Re prod uced wi th perm iss ion from ref. 2 . 2000The Rockefeller University Press.


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36 Kano, Takenaka, and Murata

o*->o-t->o

100806040200

intactH punctateD dispersedx

cont SS BL PD SB BL+PDFig . 3. Effect ofprotein kinase inhibitorson the Golgi disassembly. Semi-intact MDCK-GTcells were incubated with mitoticXenopus egg extractsandAT P containingnoinhibitor (control, cont), staurosporine (SS), butyrolactone1 (BL), PD98059 (PD), SB203580 (SB),orBL+

PD, respectively, at 33C for 80 min. The cells were fixed, and morphometric analysiswasperformed. Reproduced with permission from ref.2 2000 The Roclcefeller University Press.

3 5 Biochemical Analysis for the Golgi Disassembly3.5.1. Kinase Inhibitors

1. Incubate 1 MKC l-washed, semi-intact MDC K-G T cells with mitotic Xenopus extract,ATP-regenerating system, GTP,andglucosein thepresenceofprotein inhibitors,25\i Mstaurosporine (protein kinase inhibitor),20 \xM SB203580 p38mitogen-activated protein kinase inhibitor),50[iM PD 98059 (m itogen-activated protein kinase kinase [M EK]inhibitor),30\JiMbutyrolactone1(cdc2 kinase inhibitor)at33Cfor 80 min.

2. Fix thecells with1 formaldehyde andviewbyconfocal microsco py.3. Countthenumber ofcellsat stages 1to III andcalculate thepercentage ofcellsateach

stage Fig.3).3 5 2 Immunodepletion of inasesFrom Mitotic Xenopus Extracts

1. Incubate 20 |J,Lmo use anti-cdc2 antibody and 30 |iL protein G-Sepharose or 30|a.Lofrabbit anti-MEK serum and 30|xLofprotein A-Sepharose at4Cfor 1hwith rotation.2. Wash ant ibody-coupled beads wi th TB containing protease inhibi tors 25 (ig/mLchymostatin, pepstatinA,leupeptin,andantipain) three tim es.3. Incubate 150 jiL of mitotic Xenopus egg (4.5-5.5 mgprotein s/m L) ex tracts with antibody-coupled beadsat4Cfor1h with rotation.4. Remove thebeads by centrifugation (230 0^ or 5000 rpm, 4C, 30 s) andtransfer thesupernatant to anew tube.5. Addfresh antibody-coup led beads to thesupernatant andincubate at 4C for 1 h withrotation.6. Repeat steps4and5twiceinpreparationofcdc2-depleted mitoticXenopus extracts.7. Remove the beads. Store the supernatant (cdc2- or MEK-depleted mitotic Xenopusextracts)at-8 0 C .8. Check the depletionofcdc2orMEK from mitoticXenopus extractsbyWestern b lotting.9. Incubate IM KC l-washed, sem i-intact MD CK -GT cells with cdc2- or MEK-depleted

Xenopus extract, ATP-regenerating system, GTP,andglucoseat33Cfor 80 min.


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Rec onstitution of Mitotic G olgi Disassem bly 363100-

^ 80-o_ 60-S 40-1 1

0 _ Jim,

1- i n

1 .si1

intacts

7;\ i i

punctatedispersed

1mock cdc2 mock MEK(C) dep. (M) dep.

Fig. 4. Inhibition of the Golgi disassembly by cdc2 - or M EK -depleted Xenopus egg extracts.Mock, cdc2- or ME K-depletedXenopus 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 MEK-depleted extracts did so at stage I (intact). Reproduced with permission from ref. 2. 2000The 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 m icroscopy.11. Co unt the numb er of cells at stages I to III and calculate the percen tage of cells at eachstage (Fig. 4).

From the resu l t s us ing the Golg i d i sassembly assay based on the semi - in tac t ce l lsys tem, Golg i d i sassembly i s d i s sec ted in to two sequen t i a l e l ementary s t eps , morpholog ica l ly and b ioch em ica l ly (F ig . 5 ) . The G olg i appara tus ap pears as a r ibbo n l ike s t ructure at the apical s ide of the cel l but then fragments to form punctate s t ructures in aM E K -d ep en d en t m an n e r ( s t ag e I t o I I ) , f o l l o w ed b y d i s p e r s i o n o f t h e G o l g i m em brane s and re loca t ion to the ER in a cd c2 -de pen den t m ann er ( s t age I I to I I I ) .


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364 Kano, Takenaka, and Murata4. Notes

1. M PF (M phase-prom oting factor) activity varies between extracts. It can be measured byassaying phosphorylation of histone HI (9,10).2. The permeab ility of the cells can be altered by various experimental con ditions: cell density, temperature, incubation time, cell type, and so on. It is important to maximizepermeabilization before starting the experiments.

3. W e use propidium iodide to check the permea bility of the cells. Propidium iodide is acell-imperm eable dye that stains nucleic ac ids and can be excited with either mercury orxenon 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. Otherimpermeable dyes or fluorescence-labeled proteins, such as trypan blue, eosin, azure A,or tetramethylrhodamine isothiocyanate isomer R (TRITC)-labeled phalloidin, can beused to check the permeab ility of the cells(1,14.15). Leakag e of endogene ous cytosol canbe 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 reseale d wi th Ca^+ as dem onstrated in refs . 16 and 17.By resealing pores after exposure of semi-intact cells with nuclear extracts and cytosolprepared from different cell types, the properties and fates of cells could be altered toother ones 17,18).5. Bec ause the assay is performed in 20 |xL reaction mixture, we need to remo ve the TBfrom the membranes completely before incubation with the reaction mixture. We usuallyplace a fragment of the membrane on a Kimwipe for a short time to absorb the TB andthen transfer it on parafilm before quickly applying the reaction mixture. To avoid dryingduring the incubation at 33C, it is necessary to cover the fragments with the lid of theculture dish. In addition, we place small pieces of wet K imw ipe under the cover to minimize moisture loss.

References1. Ka no, P., Takenaka, K., Yam am oto, A., Nagay ama, K., Nishida, E., and Murata, M. (2000)MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells bythe mitoticXenopiis extracts. J. Cell Biol. 149, 357-368.2. Ka no, P., Sako, Y., Tagay a, M., Yanag ida, T., and M urata, M. (2000) Reconstitution ofbrefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact Chinese hamster ovary cells. Mol. Biol. Cell. 11,3073-3087.3. Ka no, P., Naga yam a, K., and Murata, M. (2000) Reconstitution of the Golgi reassemblyprocess in semi-intact MDCK cells.Biophys. Chem. 84, 261-268 .4. Bh akdi, S., Tranum -Jensen, J., and Sziegoleit, A. (1985) M echanism of membrane damage by streptolysin-O. Infect. Immunol. 47, 52-60 .5. Bh akdi, S., W eller, U., W alev, I., M artin, E., Jona s, D. , and Palme r, M. (1993) A guide to theuse of pore-forming toxins for controlled permeabilization of cell membranes. Med. M icro

biol. Immunol. (Bed.) 182, 167-175.6. Lafont, P., Burkhardt, J. K., and Simo ns, K. (1994) Involvem ent of microtubule motors inbasolateral and apical transport in kidney cells. Nature 372, 801-803.7. Pimp likar, S. W., Ikonen, E., and Sim ons, K. (1994) Basolateral protein transport in streptolysin O-permeabil ized MDCK cells .J. Cell Biol. 125, 1025-1035.8. W endland, M. and Subram ani, S, (1993) Cytosol-dependent perox isomal protein importin a permeabilized cell system. J. Cell Biol. 120,675-685 .


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Reco nstitution of Mitotic Go lgi Disassemb ly 3659. M urray, A. W . and Kirschner, M . W . (1989) Cyclin synthesis drives the early em bryoniccell cycle.Nature 339,2 75-280 .

10 . Chen, R. H. and M urray, A. W. (1997) Characterization of spindle assembly chec kpoint inXenopus egg extracts.Methods Enzymol. 283,572-584 .

11. Shorter, J. and Warren, G. (2002) Golgi architecture and inheritance.Annu. Rev. Cell Dev.Biol. 18, 379-420.

12 . Rossane se, O. W. and Glick, B. S. (2001) Deco nstructing G olgi inheritance. Traffic 2,589-596.13 . Schnaar, R. L., W eigel, P. H., Kuhlenschmidt, M . S., Lee , Y. C , and Rosem an, S. (1978)Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with A'^-acetyl-glucosamine. /. Biol. Cliem. 253,7940-7951 .14. Ahnert-Hilger, G., Bha kdi, S., and Gratzl, M. (1985) Minim al requirem ents for exocy to-

sis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin. /. Biol.Chem.260, 12,730-12,734.15 . Ahnert-Hilger, G., Bader, M. F., Bha kdi, S., and Gratzl, M. (1989) Introduction of macro-molecules into bovine adrenal medullary chromaffin cells and rat pheoch rom ocytom a cells(PC12)by perme abilization with streptolysin O : inhibitory effect of tetanus toxin on catecholamine secretion.J. Neurochem. 52, 1751-1758.16. W alev, I., Bha kdi, S. C , Hofmann. F., et al. (2001) Delivery of proteins into living cellsby reversible membrane permeabilization with streptolysin-O. Proc. Natl. Acad Sci. USA98 ,3185-3190 .17. Hakelien, A. M., Landsverk, H. B., Rob l, J. M., Sk alhegg, B . S., and CoUas, P. (2002 )

Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat. Biotechnol.20, 460-466.18 . Gaustad, K. G.. Boquest, A. C , And erson, B. E., Ge rdes, A. M ., and Collas, P. (2004)Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes.

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25 Reconstitution of Golgi Disassembly by Mitotic Xenopus Egg Extract in Semi-Intact MDCK Cells

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Transcript of 25 Reconstitution of Golgi Disassembly by Mitotic Xenopus Egg Extract in Semi-Intact MDCK Cells