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Correspondine-mail mc1151Current addrPutra Malaysi

& 2007 Elsevdoi:10.1016/j

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Morphology i

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Protist, Vol.

ORIGINAL PAPER

Asymmetric Cell Division as a Route to Reductionin Cell Length and Change in Cell Morphology inTrypanosomes

Reuben Sharmaa,1, Lori Peacockb, Eva Gluenzc, Keith Gullc, Wendy Gibsonb, andMark Carringtona,1

aDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UKbSchool of Biological Sciences, University of Bristol, Bristol BS8 1UG, UKcSir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

Submitted June 8, 2007; Accepted July 14, 2007Monitoring Editor: Larry Simpson

Abstract

African trypanosomes go through at least five developmental stages during their life cycle. Thedifferent cellular forms are classified using morphology, including the order of the nucleus, flagellumand kinetoplast along the anterior—posterior axis of the cell, the predominant cell surface moleculesand the location within the host. Here, an asymmetrical cell division cycle that is an integral part of theTrypanosoma brucei life cycle has been characterised in further detail through the use of cell cyclestage specific markers. The cell cycle leading to the asymmetric division includes an exquisitelysynchronised mitosis and exchange in relative location of organelles along the anterior—posterioraxis of the cell. These events are coupled to a change in cell surface architecture. During theasymmetric division, the behaviour of the new flagellum is consistent with a role in determiningthe location of the plane of cell division, a function previously characterised in procyclic cells. Thus,the asymmetric cell division cycle provides a mechanism for a change in cell morphology and also anexplanation for how a reduction in cell length can occur in a cell shaped by a stable microtubule array.& 2007 Elsevier GmbH. All rights reserved.

Key words: Trypanosoma brucei; asymmetric cell division; differentiation; epimastigote.

Introduction

The cellular form of kinetoplastid protists, such asTrypanosoma brucei, is defined by the relativeposition of three structures: the nucleus, the singleflagellum and the kinetoplast which contains theconcatenated mitochondrial DNA. Variation of the

g author; fax +44 1223 766002@cam.ac.uk (M. Carrington).ess: Faculty of Veterinary Medicine, Universitia, 43400 UPM, Serdang, Selangor, Malaysia.

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relative position of these three structures along ananterior posterior axis is used to classify the cellmorphological types (Hoare and Wallace 1966).In the trypomastigote form, the kinetoplastlies between the posterior pole and the nucleuswhereas in the epimastigote it is positionedbetween the nucleus and anterior pole (Vickermanet al. 1988). The site of emergence of the flagellumfrom the cell body is close to the kinetoplastand consequently trypomastigotes have a longer

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flagellum than epimastigotes as the flagellumoverhangs the anterior pole of the cell (Vickermanet al. 1988).

The shape of trypanosome cells is formed by acorset of closely packed subpellicular microtu-bules (Angelopoulos 1970) that are linked to eachother and to the plasma membrane (Hemphill et al.1991). The only hole in the array of subpellicularmicrotubules occurs at the point at which theflagellum emerges from the cell body (Hemphillet al. 1991). Individual microtubules within thesubpellicular array are stable with little turnoverand the cytoskeleton is remarkably constantthroughout the cell division cycle (Sherwin andGull 1989a, 1989b). The differentiation from onecellular form to another occurs without breakdownof the subpellicular microtubule array. This raisesthe question of whether the new cell morphologycan arise by remodelling of an existing single cellor is achieved as part of a division process. Thecytoskeleton also has to accommodate growthduring proliferative cell cycles when there isan increase in both circumference and length(Robinson et al. 1995). The appearance of newmicrotubules between old microtubules allows thespacing between the subpellicular microtubules toremain relatively constant as the cell circumfer-ence increases (Robinson et al. 1995; Sherwinand Gull 1989b). The increase in cell length isdependent on the extension of microtubules andthis occurs mainly at the posterior end of the cell(Robinson et al. 1995).

On infecting a tsetse fly, the mammalian blood-stream trypomastigotes undergo a well charac-terised differentiation that results in a proliferativepopulation of procyclic trypomastigotes in theinsect midgut (Szoor et al. 2006 reviewed inMatthews 2005). Bloodstream form trypanosomesexpress a variant surface glycoprotein as thedominant cell surface molecule but, during differ-entiation, the VSG is lost and procyclic cellsexpress procyclins (Roditi et al. 1987, 1989). Inaddition, procyclics have increased cell dimen-sions (Vickerman 1985), a different pattern oforganelle movement during mitosis (Robinson etal. 1995; Tyler et al. 2001) and use differentmetabolic pathways for ATP generation (reviewedin Van Hellemond et al. 2005) but retain thetrypomastigote morphology. This differentiationoccurs over a period of 10—14 h during a G1 cellcycle arrest (Ziegelbauer et al. 1990) and theremodelling of the cell is essentially completewithin one cell cycle (Matthews et al. 1995).

As the infection of the tsetse fly progresseswithin the midgut, trypanosomes move across the

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Morphology in Trypanosomes, Protist (2007), doi:10.1016/j.protis.20

peritrophic membrane into the ectoperitrophicspace and then migrate to the salivary glands viathe foregut, mouthparts and salivary ducts(Lewis and Langridge 1947; Van den Abbeeleet al. 1999; Vickerman et al. 1988) (reviewed inAksoy et al. 2003). During this migration thetrypanosomes differentiate from the trypomasti-gote to the epimastigote morphology during anasymmetric cell division (Lewis and Langridge,1947; Van den Abbeele et al. 1999). In thesalivary glands the epimastigotes attach to theepithelium through an elaboration of the flagellarmembrane (Tetley and Vickerman 1985). In addi-tion to the repositioning of the kinetoplast relativeto the nucleus, the salivary gland epimastigotesare substantially smaller than procyclic trypomas-tigotes (Van den Abbeele et al. 1999). Thepotential to infect a mammal is dependenton a further differentiation of the salivary glandepimastigote to a non-proliferative metacyclictrypomastigote form that is able to establish aninfection in a mammal when the fly takes a bloodmeal (Tetley and Vickerman 1985; Tetley et al.1987).

Here, the differentiation from midgut trypomas-tigote to salivary gland epimastigote is re-visitedusing a variety of molecular probes to enable amore detailed description of the highly asym-metric cell division cycle that starts with atrypomastigote and produces one long and oneshort epimastigote daughter cell. Analyses of theexpression of the sister chromatid cohesin com-ponent SCC1, as a marker for S- and G2 phasesof the cell cycle, and of procyclin, as a marker forthe procyclic form-specific cell surface, werecombined with morphometric and electron micro-scopical approaches. The results show that priorto mitosis during the asymmetric cell divisioncycle, the cell is in G2 and has a single kinetoplast,a state not normally found in procyclic or blood-stream trypomastigotes. The subsequent divisionof the kinetoplast is co-incident with nuclearanaphase. The rearrangement of the relativepositions of the kinetoplast and nucleus thatmarks the differentiation of trypomastigotes toepimastigotes is primarily a result of a reductionin the distance between the nucleus and theposterior of the cell, the distance between theposterior and kinetoplast is more or less constant.The division of the kinetoplast is driven byseparation of the basal bodies (Ogbadoyi et al.2003; Robinson and Gull 1991) and the lateseparation of the basal bodies during the asym-metric cell division correlates with a short newflagellum.

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Results

Validation of the Use of anti-SCC1 as aMarker for S and G2 Phases of the CellCycle

Eukaryotic chromosomes are replicated duringS-phase and the two sister chromatids areseparated during mitosis. Between these twoevents the sister chromatids are held together bycohesin, a protein complex minimally containingfour subunits: SMC1, SMC3, SCC3 and SCC1(reviewed in Nasmyth 2005). In yeast, cohesin issynthesised and assembled onto the replicatingchromosomes during S-phase (Lengronne et al.2006) and the separation of sister chromatidsduring anaphase is dependent on the proteolysisof SCC1 (Uhlmann et al. 1999). Thus, cells containSCC1 from the beginning of S-phase until ana-phase. Homologues of the four components ofcohesin were identified in the Trypanosoma bruceigenome sequence and a functional analysis of thetrypanosome SCC1 demonstrated that it is theorthologue of yeast SCC1 (Gluenz, Sharma, Gull

Figure 1. Expression profile of SCC1 during the coTrypanosoma brucei. In order to facilitate cell cycle20-deoxyuridine (BrdU) and deoxycytidine for 2 h, fixed,33258. Scale bar represents 10 mm.

Please cite this article as: Sharma R, et al. Asymmetric Cell Divisi

Morphology in Trypanosomes, Protist (2007), doi:10.1016/j.protis.20

and Carrington, unpublished results); as part ofthis work an antiserum that recognised SCC1 wasproduced (Fig. S1). Immunofluorescence usingthese antibodies was used to place SCC1expression within the trypanosome cell divisioncycle, the stage of individual cells was determinedby the nuclear and mitochondrial DNA (kineto-plast) morphology and by detection of bromo-20-deoxyuridine (BrdU) incorporation into DNA tomark cells in S-phase (Fig. 1). Cells in G1 arecharacterised by a single nucleus and singlekinetoplast (1K1N) and an absence of BrdUincorporation. The majority of these cells wereSCC1 negative (Fig. 1a), a small numberexpressed a small crescent of SCC1 (Fig. 1b)and presumably represented cells about to enterS-phase. Cells in S-phase have one kinetoplast(1K1N) which after replication of the kinetoplastDNA assumes a dumbbell shape. The two foci ofBrdU label correspond to the antipodal siteswhere the replicated minicircles are present.These cells were SCC1 positive and the strengthof the SCC1 signal was proportional to the amountof BrdU incorporation (compare Fig. 1c and d).

urse of the cell cycle in cultured procyclic formstaging, the cells were incubated with 5-bromo-and stained with anti-BrdU, anti-SCC1 and Hoechst

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Late G2 cells were 2K1N with two well separatedkinetoplasts and had not incorporated BrdU(Fig. 1e); these cells were always SCC1 positive.Cells that were 2K2N had undergone nucleardivision prior to cell division, these cells containeda few residual spots of SCC1 but were largelySCC1 negative (Fig. 1f and g). These resultsvalidated SCC1 as a marker for cell cycle stagein trypanosomes; S-phase and G2 cells wereSCC1 positive and G1 and M phase cells wereSCC1 negative.

Analysis of Procyclic Trypomastigotes fromthe Tsetse

One day after being taken up by a tsetse fly,bloodstream form trypanosomes have differen-tiated into proliferating procyclic trypomastigotesand have a range of kinetoplast and nucleusmorphologies indicating that the cells are invarious stages of the cell cycle from G1 tocompletion of mitosis (Fig. 2). The cells alreadyexhibit the typical procyclic trypomastigote pat-tern of mitosis whereby one daughter kinetoplastis present between the divided nuclei (Fig. 2e) incontrast to the bloodstream trypomastigote pat-tern where the two daughter kinetoplasts are tothe posterior side of both daughter nuclei. Thepresence or absence of SCC1 in cells withdifferent karyotypes was similar to that forcultured procyclic cells; 16% cells were SCC1

Figure 2. Procyclic trypomastigotes present in the midgHoechst 33258 and anti-SCC1. Scale bar represents 1

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negative and 84% were SCC1 positive. Thecombination of kinetoplast and nucleus morphol-ogy and presence or absence of SCC1 thusprovided a method to determine cell cycle stageof trypanosomes isolated from tsetse flies.Attempts to label DNA by feeding flies with BrdUwere unsuccessful.

After 2—3 days of infection, trypanosomes werefound in the ectoperitrophic space and movedanteriorly in this compartment to colonise theproventriculus from 4 days onwards. During thistime, trypanosomes continued to proliferate andelongated from 21.6 7 0.9 (range 16—30)mmin the posterior midgut to 24.071.5 (range19—34)mm in the anterior midgut. The resultsshowing the time course of an infection and thetrypanosome cell types present in different partsof the fly are in Figures S2 and S3 in Supplemen-tary Data. The proventriculus contained the largestrange of trypanosome cell morphologies includingall stages previously described from the foregutand proboscis (Lewis and Langridge 1947; Vanden Abbeele et al. 1999). The remainder of theexperiments concentrated on cells present in theproventriculus.

Cell Cycle Analysis of Trypanosomes fromthe Tsetse Proventriculus

Trypanosomes isolated from the proventriculuswere stained for SCC1 to determine cell cycle

ut of tsetse flies on day 1 post-infection stained with0 mm.

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stage and for DNA to determine the relativeposition of the nucleus and kinetoplast. The DNAstaining and presence or absence of SCC1enabled a sequential ordering of the various formsbased on progression of morphological change,which included all the cell morphologies andstaining patterns observed in trypanosomes iso-lated from the proventriculus. The ordering wasbased on: an increasing length of the cell, theprogression from a round to elongated nucleus,the decreasing distance between the kinetoplastand the nucleus, the progression of the kinetoplastpast the nucleus, the division of the kinetoplast,nucleus and cell, and the appearance anddisappearance of SCC1. It became clear that allthe observed cell types were from a single celldivision cycle. The stages are described belowand shown in Figure 3.

Phase

Hoechst

SCC1

K

anterior

a b c d

Phase

Hoechst

SCC1

i j lk m

Figure 3. Trypanosome cell types found in the provestained with Hoechst 33258 and anti-SCC1. White arroanterior pole of the cell. The images are ordered toproduced by a single asymmetric division. Scale bar re

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The elongation of the procyclic trypomastigotesthat occurred during anterior migration in themidgut continued in the proventriculus and wasaccompanied by a change in the shape of thenucleus, first to an oval (Fig. 3a,b) and then anelongated form (Fig. 3c,d). During this progressionSCC1 accumulated in the nucleus (Fig. 3a—d),indicating that the cells underwent S-phase duringthe elongation of the cell and nucleus. At the sametime the average length of the cell increased from24.071.5 (range 19—34) mm in the anterior midgutto 35.170.5 (range 30 to 38) mm (see Table S1 inSupplementary Data for measurements of celldimensions). All trypomastigotes with an elon-gated nucleus were strongly SCC1 positiveindicating that these cells were in S-phase ormore probably G2 (see Fig. S4 in SupplementaryData for an analysis of SCC1 expression in

e f g h

n o

ntriculus and posterior oesophagus of tsetse fliesws point to the kinetoplast and purple arrows to theshow the succession cell types leading up to andpresents 10mm.

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different cell types). No dividing trypomastigoteswere observed. As the cells elongated, thedistance between the nucleus and the kinetoplastreduced until the kinetoplast was in a juxta-nuclear position (Fig. 3c—e). The nucleus andkinetoplast then exchanged relative positions inthe cell so that the kinetoplast which waspreviously posterior to the nucleus became ante-rior (Fig. 3f—j). First, the kinetoplast was in aposterior lateral juxta-nuclear position (Fig. 3f—g)and then in an anterior lateral juxta-nuclearposition (Fig. 3h—i). Two other events coincidedwith relative movement of the kinetoplast from aposterior lateral to anterior lateral position: thekinetoplast divided (Fig. 3h—i) and the nucleusbecame SCC1-negative, indicating anaphase(Fig. 3i—j). At this stage the diameter of theposterior end of the cell was significantly largerthat the anterior end. The cell then underwentnuclear division as an epimastigote (Fig. 3j—k) toform a 2K2N epimastigote that then underwentan asymmetric cell division (Fig. 3k—l) toproduce one short (Fig. 3m) and one long(Fig. 3n) daughter epimastigote cell. The asymme-trical nature of the division is clear from thelengths of the two daughter cells: 9.3 7 0.4 (range7—13) mm and 32.470.7 (range 24—37)mm.All the daughter epimastigote cells were SCC1-negative indicating that they remained in G1whilst in the proventriculus. The fate of thelong epimastigote remains unclear but they wereoften observed with diffuse staining of nuclearDNA (Fig. 3o) possibly indicating breakdown of thenucleus. The short epimastigote was similar tounattached epimastigotes found in the salivaryglands.

The seven cell morphologies from the proven-triculus described above were present from day 5onwards during an infection and the proportions ofdifferent cell types present in proventriculi did notvary greatly over time (Fig. S5 in SupplementaryData). Thus, the differentiation to epimastigotesoccurred shortly after the proventriculus wascolonised and continued throughout the infection.In addition, the absence from the proventriculus ofboth dividing trypomastigotes and SCC1-positiveepimastigotes suggested that the proventriculartrypanosome population is not self-sustaining andrequires the continuous arrival of new trypomas-tigotes from the midgut.

Expression of Procyclins

Procyclins are the dominant macromolecule pre-sent on the surface of procyclic trypomastigotes.

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Cells isolated form the midgut and proventriculuswere stained for GPEET and EP procyclin(Fig. 4). Midgut procyclic trypomastigotes isolatedearly in an infection (days 1—3) predominantlyexpressed GPEET procyclin with EP procyclin insome cells (Fig. 4a—c). In the proventriculus,trypomastigotes expressed EP procyclin(Fig. 4d,e) but the EP procyclin began to diminishas the asymmetric cell division started withthe movement of the kinetoplast and nucleusrelative to each other (Fig. 4f,g). The shortepimastigote daughter cell did not express EPprocyclin (Fig. 4i) and there was very limitedexpression in long epimastigotes (Fig. 4h). Thegradual loss of EP procyclin during the laterstages of the asymmetric cell division cyclesupported the ordering of cell morphologiesabove. No cell in the salivary glands waspositive for either type of procyclin (data notshown).

Morphometry of the Proventricular CellCycle

Cell dimensions were derived from both phasemicroscopy images and the location of thenucleus and kinetoplast within the cell by combin-ing fluorescence images of Hoechst-stained cellswith phase overlays (Table S1 in SupplementaryData). The extension of the flagellum beyond thecell body was measured from scanning electronmicroscope images. In trypomastigotes the exten-sion of the flagellum beyond the cell bodyconstituted 14—23% of the total cell length(average, 18%; n ¼ 11). The percentage appearedto be independent of total cell length. Thechanges in cell dimensions and location of thenucleus and kinetoplast are shown in a diagram-matic form Figure 5. The most striking observationfrom this analysis is that the distance between thekinetoplast and the posterior pole of the cell isrelatively constant (Fig. 5, green to purple lines)whereas the distance between the nucleus andthe posterior pole (green to yellow lines) is greatlyreduced during the transition from trypomastigoteto epimastigote. The second observation isthat the distortion of the shape of the nucleusto an elongated form of aspect ratio 5:1 duringthe asymmetric cell division is coincident withthe elongation and narrowing of the whole celland the subsequent cell diameter (1.2mm) isless than the original diameter of the sphericalnucleus (1.6mm).

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midgut

proventriculus

anteriorposterior

0 10 20 30 40

micrometer

Figure 5. Diagram representing the changes in celldimensions and location of organelles during the cellcycle that ends with the asymmetric division and theproduction of epimastigotes.

Phase

Hoechst

GPEET procyclin

EP procyclin

Merge

Proventriculus: long trypomastigote and asymmetrical divisionMidgut procyclic tryposmastigotes

Proventriculus:

a b c d e f g h i

long and short epimastigote

Figure 4. Trypanosome cell types from the midgut and proventriculus of tsetse flies stained with Hoechst33258, anti-GPEET procyclin and anti-EP procyclin. The midgut procyclic trypomastigotes were collectedbetween day 1 and day 4 of infection. White arrows point to the kinetoplast and purple arrows to the anteriorpole of the cell. Scale bar represents 10 mm.

7Asymmetric Cell Division in Trypanosomes

Electron Microscopy of the AsymmetricDivision

Scanning and transmission electron microscopywere used to provide a more detailed view of thecell morphologies during the asymmetric celldivision cycle. Fig. 6a illustrates the large asym-metry in the dividing cell. The old flagellum isextremely long (�35mm) and overhangs theanterior end of the cell. The posterior end ofthe cell shows the characteristic broadening andthe short new flagellum has extended such that itstip is closely associated with the old flagellum.Figure 6b—e shows progression of an asymmetricdivision. At an early stage, the very short newflagellum and the old flagellum are emerging fromthe same flagellar pocket (Fig. 6b). The newflagellum then lengthens and the distancebetween the two flagellar pockets increases(Fig. 6c—e). The tip of the new flagellum remainsassociated with the lateral aspect of the oldflagellum, suggesting the presence of a flagellaconnector (Fig. 6c—e). As the new flagellum

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Figure 6. Progression of the asymmetric division viewed by scanning electron microscopy. (a) View of awhole cell with a long old flagellum (tip marked with an arrowhead) and a short new flagellum (tip marked withan arrow). (b—e) Successive stages of the division process. The cell in b is at an early stage, with the tip ofthe new flagellum just emerging from the flagellar pocket. As the new flagellum elongates, its tip (arrow)remains associated with the old flagellum, indicative of the presence of a flagella connector. In E the newflagellum has reached its maximal length and the cleavage fold is apparent. (f) Post-division smallepimastigote. Scale bars represent 1mm.

8 R. Sharma et al.

reaches its maximal length, a cleavage fold is veryapparent (Fig. 6e). Figure 6f shows the smallerpost-division epimastigote.

Transmission electron micrographs of elongatedtrypanosomes with the kinetoplast close to thenucleus showed that the diameter of the nucleuswas not much less that that of the cell with littlecytoplasm between the nuclear and plasmamembranes (Fig. 7a,b), and revealed the juxtapo-sition of the nucleus and flagellar pocket as onepassed the other (Fig. 7b). The distance betweenthe subpellicular microtubules is normal (Fig. 7aand c). A section through an asymmetricallydividing cell shows a flagella connector-likestructure (Briggs et al. 2004; Moreira-Leite et al.2001) at the tip of the new flagellum (Fig. 7c). Noexamples of kinetoplasts that were not juxtaposedto a basal body were found and, by lightmicroscopy, kinetoplasts were always positionedat the proximal end of the flagellum. Takentogether these observations indicate that the

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kinetoplast and basal body segregation werecoupled as in other life cycle stages. In theelectron micrographs, there was no indicationthat the flagella were disconnected from the cellbody except for the anterior extension and aflagellum attachment zone structure wasobserved wherever the flagellum ran along thecell body.

Epimastigotes in the Salivary Gland areSCC1-Positive

The observation that epimastigotes from theproventriculus were always SCC1-negative indi-cated that they are in G1. In contrast, populationsof epimastigotes isolated from the salivary glandscontained a percentage of SCC1-positive cells(Fig. 8). The epimastigotes could be divided intotwo types, those free in the salivary gland ductsand those attached to the epithelium. In the

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Figure 7. Thin section electron micrographs of proventricular trypanosomes. (a—b) In elongated cells thenucleus is in close proximity to the plasma membrane, and the juxtaposition of flagellar pocket and nucleusare visible. (c) Posterior end of an asymmetrically dividing cell, showing a new flagellum emerging from theflagellar pocket. The tip of the new flagellum is connected to the old flagellum (arrow). Scale bar represents500 nm.

salivary gland epimatigotes salivary gland trypomastigotes

Phase

Hoechst

SCC1

Figure 8. Trypanosome cell types found in the salivary glands of tsetse flies stained with Hoechst 33258 andanti-SCC1. White arrows point to the kinetoplast and purple arrows to the anterior pole of the cell. Scale barrepresents 10 mm.

9Asymmetric Cell Division in Trypanosomes

unattached population, 35% were SCC1-positivewhereas 90% of the attached epimastigotes wereSCC1-positive (Fig. S2 in Supplementary Data).These observations are consistent with shortepimastigotes being arrested in G1 in the proven-

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triculus and starting to proliferate once in thesalivary glands, and possibly proliferation beingstimulated by attachment. The volume of theattached epimastigotes present in the salivarygland was at least double that of the short

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epimastigotes present in the proventriculus(cellular dimensions are in Table S1 in Supple-mentary Data). It is possible that this growth is arequirement for the cells to enter S-phase but it is

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not easily determined whether the growth occursduring transit, once the epimastigote has arrivedin the salivary gland or after attachment.

Scanning Electron Microscopy ofProventricular and Salivary Gland Stages

An overview of the cell morphologies found in theproventriculus is shown in Figure 9a—d. Thepredominant proventricular trypomastigotes areelongated cells with a single flagellum (Fig. 9a).Frequently, a bead-like structure was present atthe anterior end of the cell body (Fig. 9a inset), thenature and significance of this structure isunknown. Dividing cells are highly asymmetric,with a very short new flagellum attached via its tipto the old flagellum (Fig. 9b). The small epimas-tigote daughter cells that are a product of theasymmetric division have a short flagellum(Fig. 9c—d) and frequently a characteristic protru-sion at the posterior end of the cell (Fig. 9d). In thesalivary gland, epimastigotes are attached to theepithelium via their flagellum, and these cellspossess a long posterior protrusion (Fig. 9e).Metacyclic trypomastigotes are unattached andthe posterior end of the cell assumes a bluntappearance (Fig. 9f).

Discussion

There are at least five changes in cellularmorphology during the life cycle of Trypanosomabrucei and only one of these, the differentiationfrom bloodstream trypomastigote to procyclictrypomastigote, has been investigated in detail.In this paper, the transition from procyclic trypo-mastigote to salivary gland epimastigote hasbeen investigated. This differentiation involves a

Figure 9. Scanning electron micrographs illustratingdifferent trypanosome forms found in the tsetseproventriculus and salivary glands. (a) Long trypo-mastigote. A bead-like structure of unknown naturethat is frequently observed at the anterior tip of thecell body is marked by an arrowhead; the insetshows further examples. (b) Posterior end of anasymmetrically dividing cell with a clearly visiblecleavage fold. (c—d) Short epimastigotes found inthe proventriculus. (e) Epimastigotes in the salivarygland are attached to the gland epithelium via theirflagellum. Long protrusions extending from theposterior end of the epimastigote cells are markedwith arrows. (f) Metacyclic trypomastigote. Scale barrepresents 1 mm.

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11Asymmetric Cell Division in Trypanosomes

reduction in cell length, an exchange in relativepositions of the nucleus and kinetoplast along theanterior posterior axis of the cell, that is from atrypomastigote to an epimastigote cellular mor-phology, and the loss of the predominant procycliccell surface macromolecule. In contrast to thedifferentiation of bloodstream form to procyclictrypomastigotes which occurs during a G1 arrest,the differentiation of trypomastigotes to epimasti-gotes occurs during an asymmetric cell divisioncycle. The reduction in cell length is achieved byan asymmetric cell division and apparent sacrificeof the longer daughter cell. The rearrangement ofthe nucleus and kinetoplast results from a relativemovement of the nucleus towards the posterior ofthe cell and occurs at the same time as thenuclear division that precedes the asymmetric celldivision. The loss of the EP procyclin from the cellsurface occurs around the time of the cell division.

The description of the asymmetric cell divisioncycle here is largely in agreement with earlierreports that proventricular trypomastigotes differ-entiate to epimastigotes via an asymmetric divi-sion (Lewis and Langridge 1947; Van den Abbeeleet al. 1999). The most comprehensive description(Van den Abbeele et al. 1999) used measurementsof DNA content to show that procyclic trypomas-tigotes arrive at the proventriculus in G1 arrest(2C), undergo S-phase and on completion differ-entiate into long epimastigotes whilst in G2 (4C)before undergoing an asymmetric division toproduce one short and one long daughter epi-mastigote. The details we have been able to addthrough the use of SCC1 as a cell cycle marker,analysis of procyclin expression and more detailedvisualization of morphology are: (1) the grosschange in nuclear morphology occurs duringS-phase of the asymmetric cell division cycle. (2)The exchange in relative positions of the nucleusand kinetoplast on the anterior posterior axis ofthe cell occurs during mitosis in the asymmetriccell division cycle and anaphase occurs before thekinetoplast is to the anterior side of the nucleus.Prior to mitosis the trypomastigote is a 1K1N cellin G2, whereas the kinetoplast divides during thefirst half of G2 in procyclic trypomastigotes(Woodward and Gull 1990) resulting in most G2cells being 2K1N. (3) The division of the kineto-plast is co-incident with nuclear anaphase andoccurs whilst the kinetoplast is laterally disposedto the nucleus. (4) No SCC1-positive epimasti-gotes were observed in the proventriculus. (5) Thenew flagellum emerges from the same flagellarpocket as the old flagellum and the tip of thenew flagellum appears to be attached to the old

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flagellum via a structure similar to the flagellaconnector (Briggs et al. 2004; Moreira-Leite et al.2001). (6) The exchange of the relative positions ofthe kinetoplast and nucleus is primarily a result ofa reduction in the distance between the nucleusand the posterior of the cell, the distance betweenthe posterior and kinetoplast is relatively constant.(7) EP procyclin, the most abundant cell surfacemacromolecule (Roditi et al. 1987), is lost from thecell surface during the asymmetric cell divisioncycle. (8) The long epimastigote daughter celldoes not enter S-phase as judged by the lack ofexpression of SCC1 and it is likely that the celldegenerates as many were observed with diffuseor dispersed nuclear staining.

The changes in cell morphology describedabove involved a gradual elongation of a cellfollowed by a dramatic shortening via an asym-metric division. The cellular shape of a trypano-some is defined by a helical corset of micro-tubules and elongation during proliferative growthof procyclic trypomastigotes is achieved by slidingof microtubules against one another, insertion ofnew short microtubules and by the elongation ofmicrotubules, primarily at the posterior end of thecell (Robinson et al. 1995). The progressiveelongation of midgut trypomastigote forms, from21 to 36mm by the time the asymmetric celldivision is initiated, probably occurs by the samemechanism. The alternative mechanism involvingtwisting the spiral corset to elongate the cell wouldlikely involve alterations in the spacing betweenmicrotubules and a very different helical pitch.However, the distance between microtubulesremained the same in transmission electronmicrographs of all cell types from the proventri-culus. How does the trypanosome reverse anelongation in cell form given the stability of themicrotubule corset? The asymmetric divisionachieves a shortening in length from 21 mm in theprocyclic trypomastigote to 9mm for the shortepimastigote with maintenance of the microtubulecorset. There is a second shortening in cell lengthduring the life cycle during the differentiation of‘long slender’ to ‘short stumpy’ bloodstream formtrypomastigotes (Vassella et al. 1997; Vickerman1985). However, this change in form also includesan increase in cell diameter and it is not clearwhether it is a result of an asymmetric divisionproducing a shorter daughter cell or results fromsliding of microtubules in the corset.

Initiation of growth of the new flagellum is one ofthe earliest events in the cell division cycle inprocyclic trypomastigotes (Sherwin and Gull1989a). As the cell cycle progresses, the new

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flagellum extends to about half of the length of theold flagellum, to which it remains attached via theflagella connector (Moreira-Leite et al. 2001). Atthat point, there is a rapid increase in the distancebetween basal bodies and separation of daughterkinetoplasts (Davidge et al. 2006). The basalbodies are directly connected to the kinetoplastsand the separation of basal bodies can bevisualised as separation of the two daughterkinetoplasts (Ogbadoyi et al. 2003). In procyclictrypomastigotes, the daughter kinetoplasts sepa-rate during early G2 (Robinson and Gull 1991).Initially the new flagellum emerges through thesame flagellar pocket as the old flagellum andsubsequently the flagellar pocket divides so thateach contains a single flagellum (Moreira-Leiteet al. 2001). The length of the new flagellum has aprofound effect on the location of the cleavage inthe subsequent cell division. In procyclic cells thenew flagellum can be made shorter than usual byinhibiting intraflagellar transport using RNAi andthe subsequent site of initiation of cleavage isaltered with the result that there is an asymmetricdivision (Kohl et al. 2003). One of the most notablefeatures of the asymmetric division that occurs inthe proventriculus is the disparity in length of thenew and old flagellum. The behaviour of the newflagellum is similar to that in procyclic cells; itemerges from the same pocket as the oldflagellum and the tip remains attached to the oldflagellum, probably via a flagella connector. Therestricted length of the new flagellum couldprovide the spatial information that positions theasymmetric division plane.

By the time that mitosis and cytokinesis occursin proliferating procyclic trypomastigotes the newflagellum is almost as long as the old flagellum(Robinson et al. 1995) and there is only limitedgrowth of the new flagellum in the daughter cell. Inall proventricular stages measured, the flagellumextended beyond the anterior pole of the cell by15—20% of the total flagellum length. During theasymmetric cell division cycle the trypanosomeelongates by 6 mm between the onset of S-phaseand cell division, this is accompanied by contin-uous elongation of the flagellum throughout thecell cycle, so the elongation of the cell body isaccompanied by a proportional growth in theflagellum. The flagella of many unicellular organ-isms, such as Chlamydomonas reinhardtii, areephemeral and are reabsorbed at cell division andcan also vary in length depending on parameterssuch as component availability. In other species,such as trypanosomes, the flagella is maintainedthroughout the cell cycle and the usual situation is

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either possession, or not, of a flagellum of adefined length. Hence, this demonstration of theability of a trypanosome to control the length of itsflagellum, first in the production of a long flagellumin the progenitor cell and second in the formationof the short flagellum in the asymmetric division,defines a new level of flagellum length controlhitherto unreported.

Once in the salivary glands the epimastigotesattach to the gland epithelium via an elaboratedflagellum membrane (Vickerman et al. 1988). Theattached epimastigotes proliferate whilst remain-ing attached to the epithelium (Vickerman et al.1988). The repositioning of the basal body to theanterior half of the cell, nearer to the origin of celldivision cleavage, may facilitate cytokinesis intrypanosomes attached to a substratum throughthe flagellum. Other kinetoplastids that attach toepithelia in their hosts, such as the promastigotesof Leishmania spp., epimastigotes in other trypa-nosome species and the insect-infecting Crithidiaspecies, also have basal bodies in the anteriorpart of the cell. In contrast, there are no reportedexamples of trypomastigotes that proliferatewhilst attached. It is a puzzle why trypanosomesrearrange the position of their organelles andanterior positioning of the basal body may benecessary to allow division of kinetoplastid cellsattached by a flagellum.

The loss of EP procyclin during the asymmetriccell division cycle was not unexpected as it hasbeen reported that the protein is absent in salivaryglands (Urwyler et al. 2005) and more recently ithas been shown that the predominant proteins onthe surface of epimastigote forms are a family ofalanine rich proteins previously known as BARP(Urwyler et al. 2007). The high level of expressionof procyclins is an adaptation to the tsetse midgut,the epimastigote is adapted for migration to andcolonisation of the salivary glands. It would beinteresting to know whether the composition ofthe membrane on the new flagellum was the sameas the old or included additional proteins neces-sary for the membrane elaboration and attach-ment that occurs in the salivary glands (Vickermanet al. 1988).

Some unexpected observations were madeusing scanning electron microscopy of wholecells. The elongated trypomastigotes had a dis-crete bead-like structure at the anterior end of thecell body (Fig. 9a) and the attached epimastigoteshad a spike-like protrusion at the posterior end,both in the proventriculus (Fig. 9c,d) and, in amore exaggerated form, facing away fromthe epithelium in the salivary gland (Tetley and

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Vickerman 1985) (Fig. 9e). Both these structuresremain of unknown function but it is tempting tospeculate that the protrusion in the epimastigote,which exposes a greater surface area of plasmamembrane to the gland lumen, may be involved insome interaction with signal from or to theenvironment.

Here, we have provided a more detaileddescription of an asymmetrical cell division cyclethat is an integral part of the Trypanosoma bruceilife cycle. The cell cycle includes an exquisitelysynchronised exchange in organelle locationalong the anterior posterior axis and asymmetricdivision that are coupled to a change in cellsurface architecture. The behaviour of thenew flagellum is consistent with a role in determin-ing the location of the plane of cell division,previously characterised in procyclic cells. Theasymmetric division may represent a mechanismto produce a large reduction in cell length in a cellshaped by a stable microtubule array. It nowremains to determine how the changes in cellmorphology are reversed when the epimastigotedifferentiates back to a trypomastigote in thesalivary glands.

Methods

Trypanosomes: Procyclic forms of Trypanosoma bruceibrucei Lister 427 were cultured in SDM-79 medium (Brunand Schonenberger 1979). Male tsetse flies, Glossina morsi-tans morsitans, were infected 24—48 h post-eclosion withbloodstream form T. b. brucei J10 (MCRO/ZM/74/J10[clone 1]) in sterile defibrinated horse blood containing 60mM N-acetyl glucosamine and 4�106 trypanosomes/ml.Maintenance feeding of flies was on sterile horse blood.

Dissection: Flies were starved for 48 h before dissectionwhere possible to avoid contamination with blood. Wholetsetse alimentary tracts, from the proventriculus to the rectum,were dissected in a drop of PBS (137 mM sodium chloride,2.7 mM potassium chloride, 10.1 mM di-sodium hydrogenphosphate, 1.8 mM potassium di-hydrogen phosphate pH 7.6)at various time points during the course of the infection; dailyfrom day 1 to day 10 post infection, and subsequently everyalternate day until day 28 or day 35 depending on theexperiment. Sections of the gut (proventriculus and posterioroesophagus, anterior midgut, posterior midgut) and salivaryglands were placed separately in 50ml drops of PBS onpolylysine coated slides. The various gut segments wereteased apart with 26 gauge needles to release the trypano-somes. The trypanosomes were fixed in 2% paraformalde-hyde and processed for immunofluorescence microscopy. Tocollect migratory forms from the oesophagus and mouthparts,flies were allowed to probe onto warm, alcohol-cleanedmicroscope slides prior to feeding, having received their lastmeal 48 h previously (Lewis and Langridge 1947). The drieddrops of salivary exudate were rehydrated by immersing theslides in PBS as soon as possible and then processed asabove.

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Production of anti-SCC1: The gene Tb927.7.6900 (http://www.genedb.org/genedb/tryp/) was identified as a putativeorthologue of SCC1 using BLAST searches. The open readingframe was amplified using the oligonucleotides: 50-AAG-CTTCCGCCACCATGTTCTACTCAACCTAC and 50-TGATCAA-GAACCCACAGTGAGTTGCACCTC. The subsequent HindIIIBclI fragment was cloned into the HindIII and BclI sites ofp2171, a derivative of pET15b. The recombinant SCC1 wasexpressed in E. coli BL21 (DE3) and was insoluble. It waspurified by preparative SDS-PAGE and used to immunise onerabbit. The anti-SCC1 was affinity purified using Western blotstrips containing recombinant SCC1.

Light microscopy: Cultured procyclic form trypanosomeswere washed with vPBS (137 mM sodium chloride, 2.7 mMpotassium chloride, 16 mM sodium phosphate 3 mM potas-sium di-hydrogen phosphate, 46 mM sucrose, 10 mM glu-cose, pH 7.6) and were fixed in an equal volume of 4%paraformaldehyde in PBS for 15 min on ice. Nonidet NP-40was added to a final concentration of 0.1% and the cellsincubated at room temperature for 10 min. The cells wereallowed to settle on polylysine-coated slides for 15 min in ahumid chamber. Slides were washed as follows: 2� 5 min inPBS, 2� 5 min in 0.5 M glycine, 2� 5 min in PBS, thenincubated for an hour with blocking solution (10% normaldonkey serum in PBS) in a humid chamber. Slides wereincubated with affinity purified primary antibody in blockingsolution for 1 hour at room temperature, then washedfor 3� 5 min in PBS. Slides were incubated with Alexafluor488-conjugated donkey anti-rabbit or Alexafluor 594-conju-gated donkey anti-mouse secondary antibodies diluted 1:200in blocking solution for 1 h at room temperature, then washed3� 5 min in PBS. Following the last wash, slides wereincubated with 0.1 mg/ml Hoechst 33258 in PBS for 15 min.The slides were mounted using FluorSave reagent, driedovernight at 15 1C and stored at 4 1C. The following primaryantibodies were used for immunofluorescence analyses:Rabbit anti-SCC1; anti-EP procyclin monoclonal (a kind giftof Terry Pearson); rabbit anti-GPEET polyclonal (a kind gift ofTerry Pearson). Slides were viewed using a Nikon Optiphot-2epifluorescence microscope with the appropriate filters.Digital raw grey images (8 bit and 16 bit) were taken with anattached digital camera (Photometrics Coolsnap fx), using a100� objective (Nikon Plan 1.25na oil) and RS Image version1.0.1 software (Roper Scientific, Inc). Images were laterimported into Adobe Photoshop CS and false colour wasadded using the channel mixer tool. Final images wereassembled in Adobe Illustrator CS.

Cell morphometry: Digital measurements were performedon individual cells using the morphometry software ImageJ(Research Services Branch, National Institutes of Health,Bethesda, Maryland, USA). All measurements were mm andreported as the mean 7 s.e. and ranges.

BrdU labelling: Bromo-2-deoxyuridine and deoxycytidinewere added to a final concentration of 50mM to a cultureof procyclic trypanosomes in SDM-79. After two hoursthe cells were harvested, fixed as above, and the incorpo-rated BrdU visualised using an anti-BrdU and endonucleasecocktail (GEC/Amersham) according to the manufacturersinstructions.

Electron microscopy: For scanning electron microscopy,the proventriculus and salivary glands were dissected onpolylysine coated coverslips and trypanosomes were left tosettle for at least 5 min. The cells were then fixed with 2.5%glutaraldehyde in 100 mM phosphate buffer pH 7.4 overnight,washed with water, and dehydrated through an ethanol series.The coverslips were critical-point dried and coated with gold.

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For thin section transmission electron microscopy, theproventriculus was placed directly in 2.5% glutaraldehyde,2% paraformaldehyde and 0.1% picric acid in 100 mMphosphate (pH 7.2) and processed as described previously(Dawe et al. 2005).

Acknowledgements

The authors would like to thank Terry Pearson forthe gift of antibodies, Vanessa Ferris and JennyBerry for help with tsetse fly maintenance anddissection and Mike Shaw and Sue Vaughan forhelp with electron microscopy. RS held a PhDstudentship funded by the Ministry of Science,Technology and Innovation, Malaysia. Work inMC’s WG’s and KG’s labs is funded by theWellcome Trust. KG is a Wellcome Trust PrincipalResearch Fellow.

Appendix A. Supplementary materials

The supplementary data associated with thisarticle can be found in the online version atdoi:10.1016/j.protis.2007.07.004

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