The use of nocodazole in cell cycle analysisand parasite purification

from Theileria parva-infected B cellsMartin Baumgartnera, Isabelle Tardieuxb, Hélène Ohayonc,

Pierre Gounonc, Gordon Langsleya

aUnité de biologie des interactions hôte-parasite, Institut Pasteur, 75724 Paris cedex 15, FrancebBiochimie & biologie moléculaire des insectes, Institut Pasteur, 75724 Paris cedex 15, France

cStation centrale de microscopie electronique, Institut Pasteur, 75724 Paris cedex 15, France

(Received 24 June 1999; accepted 30 July 1999)

ABSTRACT – Theileria parasites transform bovine leukocytes and induce uncontrolledlymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purifiedstage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-hostrelationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin bytaking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success toimprove quantity and quality of purified parasites. We furthermore show that nocodazole is a usefultool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells. © 1999 Éditions scientifiques et médicales Elsevier SAS

Theileria / aerolysin / parasite purification / nocodazole / Hck

1. Introduction

Theileria parva is an obligate intracellular, protozoanparasite that causes East Coast Fever, an acute leukaemia-like disease of cattle. Following invasion of T or B cells, T.parva resides as a macroschizont in the host cell cyto-plasm. The macroschizont induces its host leukocyte todivide, and the transformation process both propagatesthe parasite and clonally expands the host cell population,with often fatal consequences for the infected cattle(reviewed in [1]). Unusually for a transformed cell thephenotype can be reversed, since upon drug (hydrox-ynaphtoquinone derivative, BW720)-induced parasitedeath the leukocyte either returns to a quiescent state, ordies [2]. Theileria-transformed leukocytes can be growncontinuously in vitro, without the addition of any exog-enous growth factors and when injected into scid miceform metastatic tumors [3].

The capacity of T. parva and T. annulata to induceleukocyte transformation implies that within the infectedleukocyte these two species express ‘oncogenic’ sub-stances, and as a step towards their identification, both weand others have undertaken the study of parasite-activatedleucokyte signal transduction pathways (for recent publi-

cations see [1, 4, 6]). For example, having established thatboth species of Theileria induced the activation of thehost-cell transcription factor AP-1 [6] it is possible todevelop strategies that might lead to the isolation of theparasite gene(s) responsible. Although parasite DNA canbe readily obtained from infected erythrocytes due to theabsence of host cell nuclei, identification of putativeoncogenes requires purification of parasite-derived pep-tides and/or the construction of parasite-specific cDNAlibraries made from the transforming stage of the parasite’slife cycle. A protocol was described some years ago per-mitting the isolation of T. parva from infected lympho-cytes [7], but in our hands the parasites were rarely free ofhost cell nuclei and the yields were such that large quan-tities of infected lymphocytes were required.

When Theileria parasites have successfully invadedtheir host cell, the formation of an orderly array of micro-tubules surrounding the sporozoite can be observed thatinteracts with the parasite’s surface [8–10]. A few hourslater, the sporozoite differentiates into a multinucleatedmacroschizont and divides in such a way that part of themacroschizont is distributed to the two daughter leuko-cytes. We reasoned that the strong parasite-microtubuleinteraction might prevent efficient separation of the para-

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site from its host cell in the course of the aerolysin-basedpurification protocol described by Sugimoto [7]. There-fore, we aimed to improve that protocol by pharmacologi-cal disruption of host cell microtubules prior to cell lysis inorder to facilitate release of the parasite from the host cellcytoskeleton.

Microtubules are the primary components of mitoticspindles and are therefore essential for mitotic cell divi-sion. Moreover, in concert with actin and intermediatefilaments, microtubules organise the cytoplasma and con-trol trafficking, and their disruption leads to cell cyclearrest and loss of cellular architecture (reviewed in [11]).Various microtubule inhibitors have been described(reviewed in [12]). One of them, nocodazole [13],belongs to the group of colchicine-site-binders that inter-act with tubulin dimers, inhibit their assembly into micro-tubules, and enhance GTPase activity in the absence ofpolymerisation, and addition of nocodazole to mamma-lian cells cultured in vitro results in the loss of most of thecytoplasmatic microtubules including spindles [14]. Instudies with other apicomplexa parasites that are closelyrelated to Theileria, nocodazole had no effect on theparasite at concentrations sufficient to disrupt host cellmicrotubules [12].

An integral part of Theileria-induced transformation isthe parasite’s ability to induce host cell proliferation(reviewed in [1]). Studies on how the parasite induces hostcell proliferation involve cell cycle analysis, and clearresults have been difficult to obtain due to incompletesynchrony in the established cell lines. We thereforedecided to address the problems of microtubule associa-tion and absence of cell cycle synchrony through the useof compounds that dissociate the microtubule networkand lead to cell cycle arrest, as these are known to beactive on Theileria-infected leukocytes [15].

2. Cell culture

TpM409.B2 is a T. parva Muguga-infected B-cell clonedescribed elsewhere [16]. The B-cell characteristics of thisline grown in our laboratory were confirmed by a combi-nation of fluorescence-assisted flow cytometry (FACS)analysis of B-lymphocyte-specific surface markers andreverse transcription (RT)-PCR (data not shown).TpM409.B2 was cultivated in RPMI-1640 supplementedwith 10% foetal calf serum, 25 mM HEPES, 4 mML-glutamin, 100 µg/mL penicillin, 100 µg/mL streptomy-cin, and 10 µM â-mercaptoethanol at 37 °C.

2.1. Cell cycle analysis

Exponentially growing TpM409.B2 cells display onlyvery limited cell cycle synchrony (figure 1) and to ourknowledge, no method is available to synchroniseTheileria-infected cell cultures. For this reason, wesearched to increase the number of cells in a particularphase of the cycle in order to study restriction pointtransitions as part of our ongoing studies on Theileria-induced lymphoproliferation. Nocodazole is known toblock cell cycle progression in G2-M through disruption ofmitotic spindles (reviewed in [12]). To analyse whether

such an effect may also be perceived in TpM409.B2 cells,we performed flow cytometric DNA content analysis ofnocodazole-treated cells. To do this, we incubated 5 × 105

cells for 18 h in the presence of various concentrations ofnocodazole (SIGMA; nocodazole was made up as a10-mM stock solution in DMSO, stored in small aliquots at-20 °C and diluted to final concentration in culturemedium). Thereafter, cells were washed once in PBS andfixed overnight in 70% ethanol at -20 °C. Cells fixed insuch a way can be stored for several months in the freezer.Prior to cell cycle analysis, cellular pellets are washedtwice in PBS and resuspended in PBS containing 200 µg/mL RNAse 1A (SIGMA) and incubated at room tempera-ture for 30 min. RNAse treatment was usually performedin a volume of 100 µL which was then supplemented with400 µL staining solution [17] (3.4 mM Tris pH 7.6, 75 mMpropidium iodide, 0.1 vol/vol% NP-40, 700 U/L RNAsetype 1A (SIGMA)). Data were acquired by flow cytometryusing CellQuest software and analysed with the ModFitLTprogram for Power Macintosh. The effect of nocodazoletreatment on TpM409.B2 cell cycle distribution is shownin figure 1A. As expected, after 18 h of treatment withnocodazole at concentrations as little as 0.06 µM, most ofthe infected B cells accumulate in G2/M consistently withdisruption of host cell microtubules leading to cell cyclearrest in Theileria-infected cells. We next examinedwhether TpM409.B2 cells can re-enter cycle, such that wecould use nocodazole block release as a method to studycell cycle progression as this was described for othersystems [18]. To do this, we treated the cells with 2 µMnocodazole for 18 h and then removed the drug by twowashes in a large volume of PBS. The cells were subse-quently resuspended in fresh medium and cycle distribu-tion was measured in the following hours as describedabove. A representative flow cytometric DNA contentanalysis (figure 1B) shows that within 3 h followingnocodazole block release, between 30 and 40% of thecells synchronously re-enter G0-G1. G0-G1 exit andS-phase entry was observed 6 to 9 h after entry intoG0-G1, showing that the cells that have been releasedfrom the block are indeed capable of progressing throughthe cell cycle.

Note: Nocodazole can form crystals in culturemedium. It is very important to remove these by carefullywashing the cells in PBS prior to addition of fresh medium.For other cells, the nocodazole concentration needs to beadjusted by testing various concentrations of the drugfollowed by cell cycle analysis. Important criteria are theestablishment of a clear cell cycle block, low number ofcells with less than G0 DNA content and the capacity tore-enter G0-G1 after release of cell cycle block

3. Parasite purification3.1 Activation of the bacterial cytolytic toxin aerolysin

Aerolysin is a cytolytic bacterial protein toxin releasedby and purified from the Gram-negative pathogen Aero-monas hydrophilia [19] and was applied with success topurify schizonts from T. parva-infected bovine and buffalolymphoblastoid cells [7, 20]. Aerolysin binding may be

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facilitated by specific surface glycoproteins and causes theformation of holes of defined size. Phospholipid bilayerslacking glycoproteins such as liposomes or protease-treated red blood cells, however, can also serve as bindingsubstrates and are lysed by aerolysin [21]. The aerolysinused in this study was a gift of T. Buckley and was providedas a solution of 1 µg/µL proaerolysin, an inactive form thatneeds to be cleaved by trypsin to become active (proaerol-ysin and aerolysin is commercially available from PRO-TOX Biotech; for further information see under: http://web.uvic.ca/idc/protox/protox.html). The proaerolysinstock solution was diluted to 0.1 µg/µL in Tris-bufferedsaline (TBS: 20 mM Tris, pH 7.4, 150 mM NaCl) andmixed together with trypsin (SIGMA) to a final trypsinconcentration of 4 µg/mL. After a cleavage reaction for10 min at 37 °C, activated toxin was transferred to ice,where trypsin was inactivated by addition of a 10-foldexcess by weight of trypsin inhibitor (aprotinin, SIGMA).

3.2 Lysis of host cells

2 × 108 TpM409.B2 cells were prepared and grown to adenstity of approx. 1 × 106 cells/mL to minimise culturevolume. Prior to cell lysis, microtubules were disrupted bytreating the cells with 3 µM nocodazole for 4 h at 37 °C.Cells were then harvested by centrifugation with 200 g,washed once in ice-cold PBS and resuspended at 5 × 107

cells/ 0.9 mL in ice-cold 1 × HEPES buffer (10 mM HEPES,150 mM NaCl, 20 mM KCl, pH 7.4) containing 1 mMCaCl2 and transferred into Eppendorf tubes. In initialexperiments, direct treatment of infected cells with theactivated aerolysin resulted in complete destruction of thecells and did not allow parasite purification. Aerolysin isnot active at 0 °C, but can still bind to the plasma mem-brane of target cells, and incubation on ice thereforeallows the toxin to bind to the plasma membrane, butprevents rapid lysis of the host cells and subsequentdestruction of the parasite. We tested this modificationand added 10 µg activated aerolysin in 100 µL ice-coldTBS to each sample of 5 × 107 cells in 1 x HEPES buffer andincubated the tubes under rotation on ice for 30 min.Since aerolysin binds so tightly that it cannot be removedfrom the cells by washing [21], unbound toxin was there-after removed by two gentle washes (1200 rpm, 5 min, at4 °C in microfuge) in ice-cold BPS. Aerolysin-mediatedcell lysis was subsequently performed in 1 × HEPES con-taining 1 mM CaCl2 at 37 °C for 12 min, and plasmamembrane perforation was checked by trypan blue exclu-sion. Finally, the cells were carefully pelleted by centrifu-gation at 200 g, and 5 × 107 cells/mL were resuspended in1 × HEPES containing 5 mM EDTA.

Note: One must bear in mind that the optimal perme-abilisation conditions may vary as a function of host celltype, and the 12-min incubation period recommendedhere needs to be adjusted when using a different cell type.

3.3 Separation of purified schizonts by ultracentrifugation

Parasites were separated from host cell debris andnuclei by Percoll gradient centrifugation essentially asdescribed by Sugimoto et al. [7] with minor changes. Inbrief, a stock solution of Percoll (Pharmacia Biotech) wasprepared by mixing 8.5 parts of Percoll with 0.5 part of 20

Figure 1. Low concentrations of nocodazole induce reversibleG2-M block. A. Cells were treated with various concentrations ofnocodazole or solvent (DMSO) alone, and cell cycle distributionwas measured 18 h later by PI staining followed by FACS-asssisted DNA content analysis (M1 = less than G0 DNAcontent). B. G2-M cell cycle block was induced by treating thecells with 2 µM nocodazole for 18 h. Nocodazole was thenremoved and cell cycle progression was measured in the followinghours as in A. Percentage of cells in G0-G1 and S-phase followingnocodazole block release are shown. S-phase could include cellsthat have complete DNA synthesis and are about to enter G2.

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× HEPES (200 mM HEPES, 3 M NaCl, 400 mM KCl, pH7.4) and one part of 50 mM EDTA (pH 7.4). The cell lysate(1 mL) was added to 3.8 mL of this Percoll stock solutionand the volume was adjusted to 5 mL by the addition of 1x HEPES containing 5 mM EDTA, giving rise to 64.6%(vol/vol) final Percoll concentration. The Percoll-cell-lysate mixture was transferred into 14.5 mL Ultra-cleartubes (Beckmann) and carefully overlaid with a 45% Per-coll solution in 1 × HEPES, 5 mM EDTA. The mixture wasthereafter centrifuged at 26 000 rpm (85 000 g at rav) for30 min at 10 °C in an SW 41 Ti rotor (Beckmann). Duringcentrifugation, a gradient is established that separatesparasites by density from cellular debris and nuclei, andafter centrifugation, three bands are usually distinguish-able: two thick bands in the upper part of the gradientcontaining host cell nuclei and debris; the parasite accu-mulates in a rather faint band in the lower part of the tube(in the interphase of the gradient) from where it can becollected with a Pasteur Pipette. It is recommended toremove first the upper bands by aspiration in order toavoid contamination of the parasite preparation with cel-lular debris. Next, the parasites from the individualsamples were collected in a Corex tube and washed oncein a large volume of PBS and pelleted by centrifugation at5 000 rpm (HB-4 swing-out rotor, 2 800 g at rav) for10 min at 4 °C, resuspended in a small volume of PBS andprepared for further analysis. When the preparation is usedto isolate parasite DNA, we recommend repeating theultracentrifugation step in order to remove any trappedhost cell nuclei in the parasite fraction.

4. Analysis of purified schizonts4.1 Hoechst staining

A simple analysis to check the quality of purified para-sites is staining the parasite fraction with Hoechst 33258(Roche), a DNA intercalating agent that emits blue lightwhen irradiated with UV light. To do this, a small aliquot(10 µL) of the whole fraction is taken and mixed withHoechst to give a final concentration of 1.5 µg/mLHoechst. Analysis can be performed under a conventionalmicroscope equipped with a UV light source (350 nm),and parasite nuclei appear as small light-blue dots(figure 2B and 2D) easily distinguishable from intact cells(figure 2B) or trapped host cell nuclei. Insight into theintegrity of the parasite can be gained when standardillumination is partially added to the UV light. This proce-dure allows the visualisation of the parasite’s membraneand nuclei (figure 2C).

4.2 Analysis of parasite morphology by electronmicroscopy

In order to visualise any morphological changesinduced by nocodazole treatment, we compared the ultra-structure of parasites prepared as described herein withimages of purified parasites published by Sugimoto etal. [7]. To do this, parasites were fixed overnight at 4 °C in2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer,pH 7.3. After fixation, pellets were washed three times for10 minutes after low-speed centrifugation (2 000 rpm in

microfuge) in the same buffer and postfixed for 30 min in1% OsO4 at room temperature. Parasites were then pel-leted by centrifugation and mixed with an equal volume of2% type VII agar (SIGMA) in 150 mM NaCl. After solidifi-cation on ice, pellets were cut into small blocks whichwere washed three times for 5 min in distilled water,stained in 1% uranyl acetate in water and subsequentlydehydrated through a series of ascending concentratedacetone washes. Finally, the fixed and stained blocks wereembedded in epoxy resin (Epikote 812). After sectioningand counterstaining with 2% uranyl acetate and leadcitrate prepared according to Reynold’s method, observa-tions were performed on a Jeol 1010 electron microscopeoperating at 80 KV. Figure 3 shows that nocodazole treat-ment has no obvious effect on parasite morphology, para-site membrane is intact and parasite nuclei and organellesare clearly distinguishable. However, some shrinking ofthe area surrounding the nuclei could be observed and,even though unlikely, we cannot exclude nuclear shrink-ing as a consequence of the purified parasites’ undergoingapoptosis. Similar electron clear areas were also observedby Sugimoto et al. [7], even in intact Theileria-infectedlymphoblasts.

Figure 2. Purified T. parva parasites are intact and free of conta-minating nuclei. Light microscopical images showing A. Giemsastain of intact cells, B. Hoechst stain of intact cells, C. Hoechststain of purified parasites under a mixture of UV and standardillumination and D. Hoechst stain of purified parasites under UVlight. Hoechst-stained host cell nuclei are easily distinguishablefrom parasites by their size and brightness (B and D). Arrowheadsin A and B indicate parasites residing within host cells. (Magni-fication for all samples is 1000 ×).

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4.3 Detection of host cell- and parasite-specific proteinsby SDS-PAGE and Western blotting

In addition to optical visualisation of the parasite,analysis of proteins contained in the fraction can be usedto judge its purity. One of the molecules under study in ourlaboratory is the Src family kinase Hck, a protein tyrosinekinase highly expressed in T. parva-infected B cells andknown to be constitutively active in these cells (unpub-lished). The localisation of Hck in membrane and cytoso-lic compartments [22] and its high level of expression inTpM409.B2 renders it a useful host cell marker to checkthe purity of purified parasites. Moreover, it is of interest to

know whether the parasite contributes to the expression ofspecific proteins observed in total extracts or whether theyare exclusively derived from the host cell. Since detectionof Hck in the parasite fraction could indicate either hostcell contamination or the expression of a parasite-derivedprotein homologous to Hck, we additionally probed thesame extracts with antibodies directed to several otherhost cell proteins such as PI3-K, transferrin receptor andthe Src kinase Lyn (data not shown). To do this, we pre-pared protein extracts of purified parasites and ofTpMD409B.2 by resuspending the cells in Nonidet-P 40(NP-40) lysis buffer [20 mM Tris pH 7.4, 150 mM NaCl,

Figure 3. Membrane and ultrastructural compartments of purified parasites are intact. (A - C) T. parva parasites were purified fromTpM409.B2 by the method described in the text and analysed by electron microscopy. Panel B shows a dividing macroschizont. (N =parasite nuclei, bold arrowhead indicates parasite membrane, thin arrowhead points on mitochondria, S = shrinking area, bars correspondto 1 µm).

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2 mM EDTA, 10 mM KCl, 1% (wt/vol) NP-40, 1 mM phe-nylmethylsulfonyl fluoride (PMSF), 5 µg/mL Leupeptin,5 µg/mL Aprotinin, 5 µg/mL TLCK, 5 µg/mL TPCK, 10 mMNa3VO3 and 50 mM NaF] followed by incubation at 4 °Cunder rotation for 30 min. The lysate was cleared bycentrifugation in microfuge (13 000 rpm, 5 min, 4 °C),and protein concentration was determined using Coo-massie blue reagent (Pierce). The protein yield of highlypurified parasites (two rounds of Percoll gradient centrifu-gation) was approximatly 10 µg protein/1 × 107

TpM409.B2 cells compared to 500–800 µg total proteinobtained from the same cell line. 2 × SDS-loading buffer(100 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 0.2%bromophenol blue; 10% â-mercaptoethanol was addedjust before use) was then added and samples were incu-bated for 10 min at 95 °C. 25 µg of total protein wereseparated by SDS-polyacrylamid (8.0%) gel electrophore-sis and electro-transferred onto nitrocellulose membrane(Schleicher & Schuell). Western blot analysis was per-formed in 5% nonfat milk (in 10 mM Tris (pH 8.0),150 mM NaCl, 0.1% Tween-20). Immunoblots weredeveloped by using the Super Signal detection kit (Inter-chim) according to the manufacturer’s instructions andexposed to HyperFilm (Amersham Live Science). An anti-Hck-specific rabbit serum (Transduction Laboratories,dilution 1:1 500) was used to test extracts for Hck expres-sion. The absence of detectable Hck protein in the parasitefraction demonstrates the lack of contamination of puri-fied parasites with host cell membranes or cytosolic pro-teins (figure 4, upper panel). This result was corroboratedby the lack of detection of other host cell proteins in theparasite fraction as shown by Western blotting (data notshown). To check the parasite protein fraction, we used anantibody raised against T. annulata cdc2-related kinase(TaCRK2, dilution 1:500) [23] that revealed a strong bandat the correct size of 34 kDa in the parasite fraction,whereas the protein was only very weakly detectable inthe host cell (figure 4, lower panel).

5. Discussion

During the last few years, data have accumulated thathint to a close association between Theileria parasites andthe host cell microtubule network. Following invasion, forexample, the parasite associates with microtubules [8, 9,24] and one of the major tasks of microtubules inTheileria-transformed cells, as in other cell types, is theseparation of chromosome pairs during mitosis such thatdisruption of microtubules impairs cell division and resultsin growth arrest of Theileria-infected cells [15]. In thisstudy, we used the colchicine site-binder nocodazole, anagent that provokes reversible growth arrest in mamma-lian cells [13, 14] to test its capacity to induce cell cyclesynchronisation of asynchronously growing T. parva-infected B cells. We showed by measuring cell cycledistribution that microtubule disruption by nocodazolecompletely blocks transition through G2-M phase andresults in the accumulation of the large majority of thecells (> 90%) in that phase (figure 1). Accumulation inG2/M is not irreversible, since release of the block permits

re-entry into G0-G1 and progression to S-phase (figure 1).As in other systems [18], nocodazole can therefore beused to induce a partial cell cycle synchronisation inTheileria-infected cells, sufficient to study regulators ofS-phase entry and/or progression. However, since not allof the whole cell population is released from G2/M block,one has to bear in mind that such experiments need to beperformed under conditions where cell cycle distributioncan be followed with the method described above.

Additionally, we took advantage of the nocodazolesensitivity of bovine microtubules in order to improve aparasite purification protocol described by Sugimoto [7].In contrast to purification protocols described before [7,20], aerolysin directly applied at 37 °C lysed not only hostcells, but also parasites. It is likely that the aerolysin usedin this study was more effective and did not exclusivelybind to surface glycoproteins of mammalian cells, but alsoto plasma membrane lipids as has been proposed [21].Wehence allowed the toxin to bind by preincubation of thecells on ice and then removed unbound aerolysin toprevent parasite lysis. Even though short-term treatmentwith nocodazole at 37 °C may not be sufficient to disruptall microtubules [24], the combination of nocodazoletreatment and incubation on ice prior to aerolysin lysisresulted in a much greater amount of highly purifiedparasites. Nocodazole treatment and incubation on icetherefore lead to a dramatic increase in the number andquality of purified parasites and significantly reduce cul-ture volume, an important consideration when parasiteshave to be purified routinely.

Figure 4. Purified schizonts are free of host cell kinase Hck,whereas parasite-derived proteins can be accumulated. Equalamounts (25 µg) of total cell extracts from TpM409.B2 cells andpurified parasites were separated by SDS-polyacryamid gel elec-trophoresis and levels of Hck (upper panel) were determined byimmunoblotting. Membrane was then stripped and reprobedwith an antibody directed against TaCDK2 (lower panel).

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At the ultrastructural level, electron microscopic analy-sis of purified parasites revealed no damage to parasitespurified by this method, as both membranes and subcel-lular compartments appear intact (figure 3). This is consis-tent with the observation [12], that at comparable concen-trations nocodazole did not affect development andproliferation of Plasmodium falciparum, an apicomplexaparasite closely related to Theileria. Some electron weakareas (clear patches) surrounding parasite nuclei could beobserved and we interpret this as first signs of nuclearshrinking probably as a consequence of apoptosis. It is notlikely that shrinking is a result of nocodazole treatment,since similar electron-light areas can also be observed inparasites purified by protocols described earlier [7]. Asecond possibility is that they are induced during fixationof the cells, since essentially the same protocol was usedin both studies. Nonetheless, additional analysis ofHoechst-stained parasites by light microscopy (and trypanblue coloration, not shown) did not reveal any damageand confirmed the ultrastructural integrity. Moreover, itwas found to be an easy and efficient way to estimate thepurity of purified parasite preparation (figure 2).

By Western blot analysis we tested protein extracts ofpurified schizonts for expression of the host cell kinaseHck. Given its membrane and cytoplasmatic localisa-tion [22], the lack of detectable Hck in the parasite frac-tion illustrates the absence of contaminating host cellmembranes (figure 3, upper panel). Moreover, the signifi-cant increase of TaCRK2 in the parasite fraction (figure 3,lower panel) demonstrates that the purification protocoldescribed here is a powerful tool to isolate parasite pro-teins derived from the transforming stage of T. parva. Forhost cell signal transduction studies, it is important toknow whether the parasite contributes to protein levelsand hence activities of kinase and phosphatase observedin whole cell extracts. The results described here there-fore, allow us to exclude the parasite’s contribution to Hckkinase activities observed in TpM409.B2 cells (unpub-lished).

We showed that a nocodazole-induced cell cycle blockis established rapidly in T. parva-infected B cells and that itcan be reversed when the medium containing the drug isreplaced by fresh drug-free medium. Nocodazole can alsobe used to improve the quantity and quality of parasitespurified according to protocols based on aerolysin-mediated host cell lysis. Parasites purified in such a wayare intact and free of host cell membranes and nuclei, andthe preparation can be used to isolate parasite proteinsand to extract DNA and RNA. Even though the exactworking concentration of nocodazole for other Theileria-infected cell lines needs to be tested for optimal results, thepotential of microtubule dissociation by nocodazole withregard to cell cycle analysis and parasite purificationshould facilitate research on Theileria-induced leukocytetransformation.

Acknowledgments

We are grateful to Patrice Boquet for his advice and wethank Tom Buckley for generously providing us with puri-

fied proaerolysin. This work received support from theInstitut Pasteur, the CNRS and l’ARC (grant no 9294). M.B.is supported by the Swiss National Science Foundation.

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Original article Baumgartner et al.

1188 Microbes and Infection1999, 1181-1188