perturbation of phosphatidylethanolamine synthesis affects

Perturbation of phosphatidylethanolamine synthesis affectsmitochondrial morphology and cell-cycle progression inprocyclic-form Trypanosoma brucei

Aita Signorell,1†‡ Eva Gluenz,2‡ Jochen Rettig,3

André Schneider,3 Michael K. Shaw,2 Keith Gull2 andPeter Bütikofer1*1Institute of Biochemistry & Molecular Medicine and3Department of Chemistry & Biochemistry, University ofBern, 3012 Bern, Switzerland.2Sir William Dunn School of Pathology, University ofOxford, Oxford OX1 3RE, UK.

Summary

Phosphatidylethanolamine (PE) and phosphatidyl-choline (PC) are the two major constituents of eukary-otic cell membranes. In the protist Trypanosomabrucei, PE and PC are synthesized exclusively via theKennedy pathway. To determine which organellesor processes are most sensitive to a disruptionof normal phospholipid levels, the cellular conse-quences of a decrease in the levels of PE or PC,respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAiagainst ethanolamine-phosphate cytidylyltransferase(ET) disrupted mitochondrial morphology andultrastructure. Electron microscopy revealed alter-ations of inner mitochondrial membrane morphology,defined by a loss of disk-like cristae. Despite thestructural changes in the mitochondrion, the cellsmaintained oxidative phosphorylation. Our resultsindicate that the inner membrane morphology ofT. brucei procyclic forms is highly sensitive to adecrease of PE levels, as a change in the ultrastruc-ture of the mitochondrion is the earliest phenotypeobserved after RNAi knock-down of ET. Interferencewith phospholipid synthesis also impaired normalcell-cycle progression. ET RNAi led to an accumula-tion of multinucleate cells. In contrast, RNAi againstcholine-/ethanolamine phosphotransferase, whichaffected PC as well as PE levels, caused a cell divi-

sion phenotype characterized by non-division of thenucleus and production of zoids.

Introduction

Cellular membranes are complex mixtures of lipids andproteins and maintaining the specific composition of theplasma and organellar membranes is a dynamic, tightlyregulated process fundamental to the survival and prolif-eration of a cell. Lipid and protein compositions varybetween organisms, cell types and subcellular organellesand between the two leaflets of the lipid bilayer (van Meeret al., 2008). Membrane lipid composition and turnovernot only affects cell and organelle biogenesis, but alsomembrane and protein function. It has been well estab-lished that modulation of, for example, phosphatidyletha-nolamine (PE) and cardiolipin concentrations in bacterialand mitochondrial membranes, and in many model mem-brane systems, may result in misfolding or topologicalmisorientation of membrane proteins resulting in dysfunc-tional proteins (Bogdanov and Dowhan, 1999; Jensenand Mouritsen, 2004; Lee, 2004; Mileykovskaya et al.,2005). Membrane lipid abnormalities have also beenimplicated in several diseases, many of them affectingmitochondrial membrane and protein function (Bogdanovet al., 2008).

The complex tubular network of the single mitochon-drion in Trypanosoma brucei parasites represents anextreme form within a spectrum of diverse mitochondrialstructures across eukaryotes (Schneider, 2001; Embleyand Martin, 2006). It is subject to morphological and func-tional changes during differentiation between the distinctlife-cycle stages. Mitochondria in slender bloodstreamparasites in the mammalian host form a single tube withfew short cristae and there is no functional Krebs cycle.During the slender-to-stumpy transformation, a respira-tory switch occurs whereby the mitochondrion developswell-defined tubular cristae and becomes active in NADHoxidation (Vickerman, 1965; Brown et al., 1973). Trans-formation from stumpy bloodstream forms to procyclictrypanosomes is accompanied by a switch from glucoseoxidation to proline oxidation. In procyclic forms, whichproliferate in the tsetse fly midgut, a fully functional Krebs

Accepted 18 April, 2009. *For correspondence. E-mail [email protected]; Tel. (+41) 31 631 4113; Fax (+41) 31 6313737. †Present address: Department of Biochemistry, Weill CornellMedical College, New York, NY 10065, USA. ‡These authors contrib-uted equally to this work.

Molecular Microbiology (2009) 72(4), 1068–1079 � doi:10.1111/j.1365-2958.2009.06713.xFirst published online 30 April 2009

© 2009 The AuthorsJournal compilation © 2009 Blackwell Publishing Ltd

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cycle operates and the mitochondrion forms an extensivetubular network with plate-like cristae (Brown et al., 1973).Clearly, mitochondrial structure is dynamic and there arecorrelations between structure and function where proteinand lipid compositions are critical determinants. However,the regulatory mechanisms of mitochondrial morphogen-esis are not understood.

In T. brucei, PE and phosphatidylcholine (PC) togethercomprise about 70% of total membrane phospholipids(Patnaik et al., 1993). PE metabolism has been studiedmainly in respect to its role in the biogenesis of the gly-cosylphosphatidylinositol anchor used for membraneattachment of important surface proteins, such as thevariant surface glycoprotein (Rifkin and Fairlamb, 1985;Rifkin et al., 1995). Bloodstream and procyclic forms ofT. brucei differ in phospholipid subclass and molecularspecies composition (Dixon and Williamson, 1970;Patnaik et al., 1993; van Hellemond and Tielens, 2006),but little is known about the lipid composition and dynam-ics of organellar membranes, and the consequences ofperturbed lipid synthesis. Mutant phenotypes attributed tochanges in lipid composition include alterations in mito-chondrial morphology and inhibition of respiration follow-ing RNAi knock-down of the mitochondrial acyl carrierprotein (ACP) (Guler et al., 2008) and the cytokinesisdefects following RNAi knock-down of serine palmitoyl-transferase, an essential enzyme in sphingolipid biosyn-thesis (Fridberg et al., 2008).

We have recently elucidated the complete pathway forPE synthesis in T. brucei procyclic forms and demon-strated that de novo PE synthesis depends exclusivelyon the Kennedy pathway (Signorell et al., 2008a) (Fig. 1).In the present report, we use RNAi to knock downthe Kennedy pathway enzymes ethanolamine kinase(EK), ethanolamine-phosphate cytidylyltransferase (ET),ethanolamine phosphotransferase (EPT) and choline-/ethanolamine phosphotransferase (CEPT) to study inmore detail the cellular and morphological consequencesof altered phospholipid composition in T. brucei procyclicforms. By careful analysis of early-onset phenotypes, wediscovered that the most immediate effect of an RNAi-mediated depletion of ET was a change of mitochondrialmorphology and perturbation of the inner mitochondrialmembrane topology. In contrast, RNAi knock-down ofCEPT resulted in a specific cell division phenotype withproduction of anucleate zoids.

Results

Onset of phenotype

We have previously reported a detailed phospholipidanalysis of T. brucei procyclic forms after 5 days of induc-tion of RNAi against EK, ET, EPT and CEPT (Signorell

et al., 2008a,b). These compositional analyses repre-sented values for whole cells after depletion of the respec-tive enzymes after extended incubation times and, thus,may have missed possible transient changes, or specificor preferential effects on particular organelles. To studythis we have extended our biochemical analyses andcomplemented them with a detailed characterization ofthe cellular phenotypic consequences of lipid modulation.Cell-cycle progression and DNA content were analysedby microscopy-based approaches and flow cytometry,whereas mitochondrial morphology and activity werestudied by immunofluorescence microscopy andMitoTracker staining, combined with measurement ofmitochondrial ATP production. Finally, an electron micro-scopic approach was used to visualize specific organelleimpairment.

To obtain detailed information on the RNAi-mediatedphospholipid changes over time, we measured the totalphospholipid composition of all four T. brucei RNAimutants, EK, ET, EPT and CEPT, at 24 h intervals duringthe first 5 days of induction, using two-dimensional thin-layer chromatography (TLC) and lipid phosphorusdetermination. Knock-down of EK, ET and EPT resulted ina progressive decrease of the PE to PC ratio during thefirst 3 days of induction and thereafter reached a plateauat 0.21 for EK and 0.07 for ET RNAi cells (Fig. 2). These

Fig. 1. Biosynthesis of PE and PC in T. brucei procyclic forms.Schematic representation of the CDP-ethanolamine andCDP-choline branches of the Kennedy pathway (adapted fromSignorell et al., 2008a). EK, ethanolamine kinase, Tb11.18.0017;CK, choline kinase, Tb927.5.1140; ET, ethanolamine-phosphatecytidylyltransferase, Tb11.01.5730; CT, choline-phosphatecytidylyltransferase, Tb10.389.0730; EPT, ethanolaminephosphotransferase, Tb10.389.0140; CEPT, choline-/ethanolaminephosphotransferase, Tb10.6k15.1570. In this study, the expressionof EK, ET, EPT and CEPT was downregulated by RNAi.

PE affects mitochondrial morphology in T. brucei 1069

© 2009 The AuthorsJournal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1068–1079

results are consistent with our previous findings thatdownregulation of ET for 5 days causes a more severedrop in PE than RNAi against EK (Signorell et al., 2008a).A decrease in the PE to PC ratio to 0.23 was alsoobserved during downregulation of EPT (Fig. 2); however,as previously shown, this was not due to a drop in PE butrather an increase in PC (Signorell et al., 2008a). In con-trast, the ratio of PE to PC increased slightly to 0.44during RNAi against CEPT (Fig. 2), which is consistentwith the previously observed decrease of the PC contentin CEPT RNAi cells after 5 days of induction (Signorellet al., 2008a). The relative amounts of the other phospho-lipid classes, phosphatidylserine (PS), phosphatidylinosi-tol, inositolphosphoceramide, sphingomyelin andcardiolipin, remained unchanged during the entire time-course of RNAi against all four PE biosynthetic enzymes(results not shown), except that PS dropped to almostundetectable levels after 5 days of downregulation of ET(see also Signorell et al., 2008a).

All RNAi mutants grew normally during the first 3 daysof RNAi induction, but stopped dividing thereafter, with theRNAi knock-down of ET causing the strongest effect ongrowth rate (Fig. S1; Signorell et al., 2008a).

RNAi against ET affects mitochondrial morphology

To study possible effects of an altered phospholipid com-position on membrane and organelle integrity, we analy-sed parasites after RNAi against all four PE biosyntheticenzymes by fluorescence and electron microscopy. UsingMitoTracker as a marker for mitochondria, we observed apronounced change in the morphology of the mitochon-drion following RNAi knock-down of ET (Fig. 3A–E).

MitoTracker staining of the uninduced control cells showsthe characteristic tubular structure of the single mitochon-drion (Fig. 3A and B). Starting at 3–4 days post induction,an increasing number of parasites showed a less complextubular pattern and, at 5–6 days post induction, theMitoTracker-positive areas in all cells were reduced to afew discrete spots (Fig. 3C–E). Because MitoTrackerstaining depends on a mitochondrial membrane potential,antibody labelling of heat-shock protein 60 (Hsp60) wasused as an alternative probe to examine mitochondrialmorphology, and this produced a staining pattern similarto MitoTracker (Fig. 3F).

Further examination of cellular ultrastructure by trans-mission electron microscopy (TEM) clearly showed abnor-mal mitochondrial morphology in ET RNAi cells (Fig. 4).The most obvious difference between control andET-depleted cells was a time-dependent disappearanceof normally shaped cristae (Fig. 4A–D). After 5 days ofRNAi, the mitochondrion was essentially devoid of theplate-like cristae characteristic of the procyclic-form mito-chondrion. Instead, numerous intramitochondrial vesicu-lar structures accumulated (Fig. 4B–D, indicated by thewhite arrows). In addition, the region of the mitochondrionharbouring the kinetoplast DNA appeared dilated(compare Fig. 4A with Fig. 4B–D). However, ET RNAi hadno obvious effect on the structure of the kinetoplast DNAdisk itself, nor did it affect its close association with themitochondrial membrane near the base of the flagellum.Interestingly, with increasing induction time the mitochon-drial matrix appeared more electron dense, suggestingchanges in matrix structure and/or composition. In addi-tion, in most cells showing abnormal mitochondrial mor-phology, we noticed the appearance of a gap between thetwo membranes of the nuclear envelope (Fig. 4D, indi-cated by the black arrow).

In contrast to the results obtained with ET RNAi cells,trypanosomes depleted of EK, EPT or CEPT using RNAishowed normal mitochondrial structure as assessed byTEM, MitoTracker staining and labelling of Hsp60 (datanot shown).

Furthermore, examination of membranes in the differ-ent RNAi cells by TEM showed no obvious morphologicalabnormalities in the Golgi cisternae, endoplasmic reticu-lum, flagellar pocket or plasma membranes (data notshown).

Mitochondria in ET RNAi cells maintain ATP production

Staining of mitochondria with MitoTracker depends on anintact mitochondrial membrane potential. To confirm thatafter downregulation of ET the mitochondria maintain pro-duction of ATP by oxidative phosphorylation, we mea-sured ATP generation in isolated mitochondria usingsuccinate as substrate (Bochud-Allemann and Schneider,

Fig. 2. Effects of RNAi-mediated knock-down of PE biosyntheticenzymes on phospholipid composition. (A) Parasites were culturedin the presence of tetracycline for the indicated times, and lipidswere extracted from 4 ¥ 108 trypanosomes using organic solvents.After separation by two-dimensional TLC, all major lipid spots werescraped from the plates and quantified by phosphorus analysis.The data represent the ratios of PE to PC after downregulation ofEK (circles), ET (crosses), EPT (triangles) and CEPT (squares).The PE to PC ratios in uninduced cells remained constant (data notshown).

1070 A. Signorell et al. �


2002). In addition, we determined ATP production bysubstrate-level phosphorylation using a-ketoglutarate assubstrate (Bochud-Allemann and Schneider, 2002). Theresults in Fig. 5 show that mitochondrial ATP production

by either pathway was unchanged in trypanosomes afterRNAi-mediated knock-down of ET. In control experiments,ATP production was blocked by the addition of the ADP/ATP translocation inhibitor, atractyloside, demonstrating

Fig. 3. Changes in mitochondrialmorphology following RNAi knock-down of ET.Trypanosomes were grown in the presence oftetracycline for 0 (A), 3 (B), 4 (C), 5 (D, F) or6 (E) days. The left panels show mitochondrialabelled with MitoTracker (A–E), or with anantibody against the mitochondrial matrixprotein Hsp60 (F). The central panels showDNA stained with DAPI, and the right panelsshow a phase-contrast image of the cells.



that ATP was indeed generated in the mitochondrion.Together, the data indicate that, although the mitochon-drial structure was altered and the organelle looked frag-mented as assessed by light microscopy, it remainedfunctional in terms of energy production.

ET RNAi affects cell shape

In addition to the effects on mitochondrial morphology,RNAi knock-down of ET also resulted in distinct alter-ations of cell shape, as revealed by scanning electronmicroscopy (SEM) (Fig. 6). Many cells (up to 12% of thepopulation) had a strikingly elongated posterior end.Typical examples of such trypanosomes viewed by SEMare shown in Fig. 6B and C. The proportion of these cellsin the population increased steadily following RNAi induc-

tion without reaching a plateau (Fig. 6E). The majority ofelongated cells were at the 1K1N (one kinetoplast, onenucleus) stage of the cell cycle but the proportion of2K1N, 2K2N and 1K2N cells with elongated posteriorends increased over time from 18.8% to 35.8% of totalcells between day 5 and 8 of induction (Fig. 6F). We alsoobserved cell pairs that had elongated posterior endswhich remained connected by long membrane bridges(Fig. 6D, indicated by the arrow head), possibly indicatinga defect in the late stage of cytokinesis.

Interference with the Kennedy pathway impairs normalcell-cycle progression

To investigate the effect of the altered phospholipid com-position on cell-cycle progression and cytokinesis further,

Fig. 4. Changes in mitochondrialultrastructure following RNAi against ET.Trypanosomes were grown in the presence oftetracycline for 0 (A), 3 (B), 4 (C) or 5 (D)days and processed for TEM. The insetsshow enlarged views of mitochondrial profiles(m) and kinetoplasts (k). Ultrastructuralchanges were observed in the innermitochondrial membrane. Examples of normaldisk-like cristae are indicated witharrowheads. Following induction of ET RNAi,vesicular structures were observed(white arrows). A distended nuclearenvelope (black arrow) was seen in somecells 5 days after induction of RNAi;n, nucleus. Calibration bars, 1 mm.



we analysed nuclei and kinetoplast distribution in cellsfor 8 days after induction of RNAi against EK, ET, EPTand CEPT. Depletion of ET or EPT resulted in adecrease in the number of parasites with one kinetoplastand one nucleus (1K1N; from 80% to < 40%; Fig. 7A),while the number of cells with multiple kinetoplasts andnuclei increased from < 1% to 24–30% (Fig. 7A), indicat-ing that a large proportion of cells in the population failedto complete cytokinesis but progressed to another roundof organelle duplication. In addition, we found that knock-down of ET caused a > 10-fold increase in the number ofanucleate 1K0N cells (zoids) from < 0.5% to 5–8%(Fig. 7A). A similar increase in 1K2N cells (from < 0.5%to 8–9%) suggests that a subpopulation of cells under-went an asymmetric cell division where a 2K2N celldivided incorrectly to produce one zoid and one 1K2Ndaughter cell. Downregulation of EK had little effect onthe number and distribution of kinetoplasts and nuclei(Fig. 7A).

Fig. 5. Mitochondrial ATP production after RNAi against ET.Mitochondria were isolated from equal numbers of trypanosomesafter 5 days of culture in the absence (white bars) or presence(grey bars) of tetracycline using digitonin. Substrate-levelphosphorylation was measured by addition of a-ketoglutarate assubstrate. Oxidative phosphorylation was measured with succinateas substrate. To control for mitochondrial ATP production, thetranslocation of ADP/ATP across the inner mitochondrial membranewas inhibited by the addition of atractyloside to the reaction. Thedata represent mean values � standard deviations from threeexperiments.

Fig. 6. Changes in cell shape following RNAi against ET.A–D. Trypanosomes were grown in the presence of tetracycline for0 (A), 3 (B), 4 (C) or 5 (D) days and processed for SEM. Theposterior end of the cell is indicated by an asterisk, and the areawhere the flagellum exits the flagellar pocket, by an arrow. Thearrowhead indicates a connection between two elongated cells.Calibration bars, 1 mm.E. Trypanosomes with an elongated posterior end (defined by a> 50% increase in length) were quantified by counting at least 500cells from three separate experiments and plotted as a percentageof the total cell population. A linear trend line was fitted.F. Trypanosomes with an elongated posterior end were analysedfor their position in the cell cycle. Cells induced for ET RNAi for5–8 days were fixed and stained with DAPI as described in Fig. 3.At each time point, the numbers of kinetoplasts (K) and nuclei (N)were counted in 64–179 cells with elongated posterior end and thefrequency of each category was plotted as the percentage of thetotal cell population. 1K1N (diamonds), 2K1N (squares), 2K2N(triangles), 1K2N (crosses), cells with > 2K and > 2N (circles).



CEPT RNAi results in production of zoids

RNAi knock-down of CEPT resulted in a dramaticincrease in 1K0N cells from 0.5% to > 38%, accompaniedby a decrease in the number of 1K1N cells from > 80% to40% (Fig. 7A). A rise in the number of 1K2N cells was notobserved following CEPT RNAi. The KN profile of CEPTRNAi cells suggested that cells were undergoing cytoki-nesis in the absence of nuclear division, resulting in theproduction of one anucleate zoid and one 1K1N cell.These cells were further examined by flow cytometry tomeasure DNA content (Fig. 8A). On day 7, 37% of thecells in the population had a DNA content of < 1C(Fig. 8B). This subpopulation, which first appeared on day3 and increased sharply thereafter, likely represents thezoids. The proportion of cells with 2C DNA content sharplydecreased between days 3 and 5 after induction (Fig. 8),which correlates with the decrease of 1K1N cells from day3 (Fig. 7A). In addition, we noticed an increasing propor-tion of cells with 8C DNA content (15% on day 7) (Fig. 8).Such increased DNA content is a feature either of cellsthat contain more nuclei than normal, or of cells thatre-initiated nuclear S-phase without having gone throughmitosis. The latter scenario is more likely for CEPT RNAicells, because the analysis of DAPI-stained cells showedno accumulation of multinucleate (> 2N) cells (Fig. 7A).Taken together, the data suggest that as a consequence

of CEPT knock-down, nuclear division fails, but DNA rep-lication continues and the cells proceed with cytokinesis.

Discussion

Changes in the membrane lipid composition are expectedto have major pleiotropic effects on a cell. Here, we analy-sed the distinct phenotypes resulting from RNAi-mediatedknock-down of enzymes of the Kennedy pathway for PEsynthesis in T. brucei procyclic forms. Mitochondrial mor-phology was severely altered by RNAi knock-down of ET,which causes the strongest reduction in PE. In contrast,RNAi knock-down of CEPT, which affects primarily PCsynthesis, resulted in a nuclear division phenotype andthe generation of anucleate zoids. These results revealwhich organelles or processes are most sensitive to aperturbation of normal levels of the major phospholipids inT. brucei.

The earliest, and most striking effect of ET RNAi is seenin the mitochondrion. This mitochondrial phenotype wascharacterized by an increased electron density, alterationof inner mitochondrial membrane morphology, affectingcristae, and loss of the branched tubular networkmorphology. Following ET RNAi, we observed a reductionin the number of normal plate-like cristae accompanied bythe appearance of vesicular structures. The most parsi-

Fig. 7. Effects of RNAi against PEbiosynthetic enzymes on cell-cycleprogression and morphology.A. Trypanosomes were cultured in thepresence of tetracycline to induce RNAi for7–8 days, fixed, and DNA was stained withDAPI. At each time point, the numbers ofkinetoplasts (K) and nuclei (N) were countedin at least 500 cells after downregulation ofEK, ET, EPT and CEPT. The frequency ofeach category was plotted as the percentageof the total cell population. 1K1N (diamonds),2K1N (filled squares), 2K2N (triangles), 1K0N(open squares), 1K2N (crosses), cells with> 2K and > 2N (circles).B. Trypanosomes were fixed 6 days afterRNAi induction and stained with DAPI.Individual composites show representativephase-contrast images (left) and DNA staining(right) of cells after knock-down of EK, ET,EPT and CEPT.



monious explanation is that these membrane boundedstructures originated from the cristae. However, theprecise relationship between these vesicular structuresand the inner mitochondrial membrane, and whetherthere was continuity between them, could not be deter-mined with certainty. ET RNAi caused the sharpest reduc-tion of total levels of PE, and resulted in a decrease of PS,without affecting the cellular levels of PC (see also Signo-rell et al., 2008a). A reduction of PE (and PS) levels insubdomains of the mitochondrion may directly affect thecurvature of the membrane and its ability to form andmaintain normally shaped cristae. Alternatively, the distri-bution of membrane proteins may have been affected bychanges in lipid composition causing disruption of normalmembrane topology. Although we cannot distinguishbetween these possibilities, the specific alterations ofmitochondrial structure early after induction of ET RNAi

provide further evidence that the inner mitochondrialmembrane of procyclic T. brucei is highly sensitive to aperturbation of phospholipid metabolism. A similar obser-vation was made in a recent study about the effectsof RNAi knock-down of ACP in procyclic-form trypano-somes (Guler et al., 2008). ACP RNAi affected thestructure of the mitochondrion and reduced oxygen con-sumption, indicating that respiration had been inhibited.Changes in lipid composition, a decrease of diacyl-PEand phosphatidylinositol in the mitochondrion, werethought to be the primary cause of disrupted mitochon-drial function because they preceded the otherphenotypes. In the present study, cells induced for ETRNAi continued to produce ATP, indicating that the elec-tron transport chain remained fully functional in thesecells, despite the ultrastructural changes. Concentrationof MitoTracker staining to discrete foci indicates thatenergetically active areas of the mitochondrion weredispersed. Whether the mitochondrion is physically frag-mented could not be determined with certainty.

Following ET RNAi, the mitochondrial profiles observedin the TEM appear more electron dense than in the unin-duced cells. The opposite effect, i.e. a reduction in elec-tron density in the mitochondrion, has been reported afterRNAi knock-down of ACP (Guler et al., 2008). In prepa-ration for TEM, cells in both studies were post-fixed withosmium tetroxide, which reacts with lipid moieties, therebyadding density to the biological material. In the ACP RNAicells, the reduced electron density may reflect a decreasein lipid content. In the ET RNAi cells, the mitochondrialmatrix appears more osmophilic, which may indicate alocal increase in lipid content.

The precise three-dimensional topology of inner mito-chondrial membranes in general has remained controver-sial and theoretical models as to how the cristae structuremight be determined at the molecular level have recentlybeen reviewed (Zick et al., 2009). Electron tomography ofrat liver mitochondria has started to provide new insightsinto mitochondrial structure and dynamics, by producinghigh-resolution models of the mitochondrial membranearchitecture and describing the tubular junctions betweencristae and inner mitochondrial membrane under differentconditions (Frey and Mannella, 2000; Mannella, 2006;2008). Dissecting the mechanisms that govern the normalmorphological changes in the trypanosome mitochon-drion, such as the correlation of cristae development witha respiratory switch to oxidative metabolism, mightuncover important general principles of mitochondrialbiology.

In addition to the mitochondrial phenotype, knock-downof ET RNAi led to the accumulation of 20% of cells with> 2 kinetoplasts and > 2 nuclei, suggesting that these cellshad failed to go through cytokinesis. Moreover, some cellsremained connected at their posterior ends by membrane

Fig. 8. Flow cytometric analysis of DNA contents in CEPT RNAicells.A. CEPT RNAi was induced for 0–7 days as indicated. At each timepoint, cells were stained with propidium iodide and subjected toFACS analysis. Each histogram represents 10 000 events.B. The data from (A) showing trypanosomes with 2C, 2–4C, 4C,8C and < 2C DNA content were plotted relative to the total cellpopulation. The gates used for quantification are shown in (A).



bridges, indicating a block or delay in the late stage ofcytokinesis. While PE is predominantly found on the cyto-plasmic side of cellular membranes, studies of mamma-lian cells have shown that re-distribution to the surface ofthe cleavage furrow is necessary for cytokinesis, andmutant Chinese hamster ovary cells with decreased PElevels exhibited a defect in late cytokinesis, which wasattributed to a defect in contractile ring disassembly(Emoto and Umeda, 2000; Emoto et al., 2005). Interest-ingly, a mutant strain of Escherichia coli deficient in PE isalso blocked in cytokinesis (Rietveld et al., 1997). ThisE. coli strain shows an absolute dependence on divalentcations for normal growth (DeChavigny et al., 1991).Since divalent cations are known to induce non-bilayer-phase formation in cardiolipin model membranes, the lackof the non-bilayer lipid PE in mutant E. coli may directly beresponsible for a block in cytokinesis (Rietveld et al.,1993; 1997). Non-bilayer lipids have been implicated inprocesses such as membrane fusion, transbilayer move-ment of lipids and membrane protein function (Siegel andEpand, 1997; van den Brink-van der Laan et al., 2004;Epand, 2007). Similarly, in T. brucei, a lack of PE maydirectly affect cytokinesis by preventing membrane fusionevents late in cytokinesis, resulting in trypanosome pairsstill connected by long membrane bridges and unable tocompletely separate. However, a direct role for PE, if any,in the formation of the cleavage furrow, furrow ingressionor abscission in T. brucei remains to be determined.

Alternatively, since division of the complex T. bruceimitochondrion is intimately linked with division of otherorganelles and a mitochondrial activation has been pro-posed to play a determining role in the morphogenesisof the procyclic form (Vickerman, 1965), disruption ofthe mitochondrial structure could have downstreameffects and possibly interfere with cytokinesis and cellmorphogenesis.

Superficially, the morphological phenotype observed inthe ET RNAi cells bears some resemblance to the nozzlephenotype defined before (Hendriks et al., 2001), which iscaused by a polar extension of microtubules at the pos-terior end of procyclic T. brucei, resulting in a specificincrease of the distance from the kinetoplast to the pos-terior end in 1K1N cells. However, in contrast to thenozzle phenotype defined by Hendriks et al. (2001), andsubsequently by Hammarton et al. (2004), we found noevidence of a G1/S block in the ET RNAi cells with‘nozzle-like’ morphology.

RNAi knock-down of CEPT produced a cell divisionphenotype characterized by accumulation of zoids. Simi-larly high proportions of zoids have previously beenobserved in T. brucei procyclic forms under conditionswhere mitosis was blocked, for example, by phar-macological inhibitors (Robinson et al., 1995; Ploubidouet al., 1999), RNAi knock-down of the mitotic cyclin CYC6

(Hammarton et al., 2003) and expression of a Separase-resistant cohesin subunit SCC1 (Gluenz et al., 2008).Zoids also accumulated under conditions where segrega-tion of post-mitotic nuclei was aberrant, following forexample treatment with the antimicrotubule agent rhizoxin(Ploubidou et al., 1999) or RNAi knock-down of centrin 4(Shi et al., 2008). Flow cytometry and cytology suggestedthat CEPT RNAi inhibited nuclear division. Our data areconsistent with a model where the first division of theaffected cells produces one zoid and one 1K1N* [4C DNAcontent (Ploubidou et al., 1999)] daughter cell. The 1K1N*cell then re-initiates a further round of organelle duplica-tion and at cell division produces another zoid and a1K1N** (8C DNA content) daughter cell. PC has previ-ously been associated with mitosis in mammalian cells.HeLa cells increase levels of PC synthesis in late mitosisand the newly synthesized PC is predominantly incorpo-rated into nuclear envelope membrane as it re-formsduring telophase (Henry and Hodge, 1983). The ortho-logue of CEPT in other organisms has been localized tothe ER and nuclear envelope membrane (Henneberryet al., 2002). Association of newly synthesized PC withthe nuclear envelope membrane is therefore not neces-sarily restricted to re-formation of a nuclear membraneafter completion of mitosis but might also occur in cellsthat undergo a closed mitosis, such as T. brucei. Trypa-nosomes probably co-ordinate membrane phospholipidsynthesis with the cell cycle, analogous to mammaliancells (Jackowski, 1994; Banchio et al., 2003), but this hasnot been demonstrated experimentally. CEPT was theonly enzyme, of the four examined in the present study,where RNAi resulted in a decrease of PC (to 64% ofwild-type levels; see also Signorell et al., 2008a). Regu-lation of nuclear division in procyclic T. brucei may thusdepend on new PC synthesis, or possibly PC levelsremaining above a threshold level. Interestingly, theknock-down of EPT caused a strong increase in PC (seealso Signorell et al., 2008a) and resulted in an increase inthe number of cells with > 2 kinetoplasts and > 2 nucleifrom 0% on day 3 to 30% on day 7 after RNAi induction.This shows that elevated levels of PC had no inhibitoryeffect on nuclear division, but cytokinesis was impaired,possibly due to a lack of PE.

In summary, we conclude that in T. brucei procyclicforms the mitochondrial structure, particularly the innermitochondrial membrane, is most sensitive to a depletionof PE levels (ET RNAi), while nuclear division is affectedby a decrease in the PC content (CEPT RNAi).

Experimental procedures

Unless otherwise specified, all reagents were of analyticalgrade and were from Merck (Darmstadt, Germany), Sigma-Aldrich (Buchs, Switzerland) or MP Biomedicals (Tägerig,



Switzerland). All EM supplies were from Agar Scientific (Stan-sted, UK) or TAAB Laboratories (Reading, UK).

Trypanosomes and culture conditions

The following gene products were downregulated byRNAi using stem-loop constructs containing a puromycinresistance gene: T. brucei EK (GeneDB Accession No.Tb11.18.0017), T. brucei ET (Tb11.01.5730), T. brucei CEPT(Tb10.6k15.1570) and T. brucei EPT (Tb10.389.0140).Details on the construction of the RNAi cell lines and ontrypanosome culture conditions are provided elsewhere(Signorell et al., 2008a,b).

Analysis of nucleus and kinetoplast configurations

Cell densities and approximate cell volumes were determineddaily using a Casy TT cell counter (Schärfe System, Reutlin-gen, Germany) or a Neubauer counting chamber. The celldensity was kept between 3 ¥ 106 cells ml-1 and 1.5 ¥ 107

cells ml-1 during all experiments. Antibiotics were omittedduring the course of the entire experiment, except for theaddition of tetracycline. At a given time point, parasites werewashed twice with phosphate-buffered saline (PBS; 137 mMNaCl, 2.6 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2)and allowed to settle onto glass microscope slides (Erie Sci-entific Company, Portsmouth, NH). Trypanosomes were fixedwith -20°C methanol for at least 10 min and subsequentlywashed twice with PBS. The slides were mounted usingVectashield containing 4′,6′-diamidino-2-phenylindol (Dapi;Vector Laboratories, Burlingame, CA) to visualize nuclearand mitochondrial DNA. Counts of nuclei (N) and kinetoplasts(K) were performed on a Leica Leitz DM RBE microscopeand on a Nikon Eclipse TE2000-E microscope. Nucleus andkinetoplast configurations were assessed from the entire cellpopulation (at least 500 cells per time point), or from thesubpopulation of cells with an elongated posterior end (64–179 per time point). Light microscopy pictures were madeusing the Leica Leitz DM RBE microscope equipped with aPhotometrics CoolSNAP fx camera.

Electron microscopy

For TEM, parasites were fixed for 5 min with 2.5% glutaral-dehyde in culture medium before being transferred to buff-ered fixative (2.5% glutaraldehyde, 2% paraformaldehyde,0.1% picric acid in 100 mM phosphate buffer, pH 7.0) for2–24 h. The fixed cells were washed with 200 mM phosphatebuffer, pH 7.0, and post-fixation was performed with 1%osmium tetroxide in 100 mM phosphate buffer, pH 7.0, for1.5 h at 4°C. After thorough washing with water, fixed cellswere stained en bloc with 2% aqueous uranyl acetate for 2 hat 4°C in the dark, rinsed with water, dehydrated with ascend-ing acetone concentrations and embedded in epon resin.Sections of 60–70 nm were analysed with a Philips Tecnai 12transmission electron microscope.

For SEM, parasites were fixed in 2.5% glutaraldehyde inculture medium for 2 h, washed and re-suspended in PBS ata density of 2–4 ¥ 107 cells ml-1. Cells were allowed to settleonto 13-mm-diameter coverslips (Chance Propper, Smeth-

wick, UK), rinsed with water and dehydrated with ascendingethanol concentrations. Samples were critical-point dried andcoated with gold. Specimens were analysed with a Jeol JSM-5510 scanning electron microscope.

Immunocytochemistry

For staining of mitochondria, 1 ¥ 107 parasites were incu-bated with 0.1 mM MitoTracker Red CMXRos (Invitrogen,Basel, Switzerland) in culture medium for 15 min at 27°C,washed twice with PBS and left to settle onto glass micro-scope slides. Cells were fixed with 4% formaldehyde in PBSfor 10 min, permeabilized with 0.1% NP40 in PBS for 5 minand washed three times with PBS. DNA was visualized byincubation with 5 mg ml-1 Dapi for 1 min. Fixed cells werewashed three times with PBS and mounted with 1%1,4-diazabicyclo[2.2.2]octan, 90% glycerol in 50 mM sodiumphosphate buffer, pH 8.0. Samples were analysed using aLeica Leitz DM RBE microscope equipped with a Photomet-rics CoolSNAP fx camera.

Immunofluorescence labelling of the mitochondrial matrixwas performed as follows: trypanosomes were washed oncewith serum-free SDM-79 and allowed to settle onto glassmicroscope slides (Erie Scientific Company, Portsmouth,NH). Parasites were fixed with 4% paraformaldehyde in PBSfor 10 min. Mouse polyclonal antiserum against mitochondrialmatrix Hsp60 was used at a dilution of 1:100 in PBS for 1 h atroom temperature. Subsequently, cells were washed threetimes with PBS and incubated with secondary antibody AlexaFluor 568 goat anti-mouse IgG (Invitrogen, Basel, Switzer-land) at a dilution of 1:1000 for 1 h at room temperature.Slides were washed three times with PBS and mounted usingVectashield. Fluorescence microscopy was performed on aNikon Eclipse E600 microscope equipped with a Nikon DigitalCamera DXM 1200 and the RCT-1® software provided by themanufacturer.

Fluorescence-activated cell sorting (FACS)

Trypanosomes (2 ¥ 107) cells were fixed in 0.25% formalde-hyde for 5 min at room temperature and subsequently in 10volumes of ice-cold 70% ethanol for 30 min on ice. Cells wereexamined by light microscopy to confirm that no clumpinghad occurred. Cells were re-suspended in 4 ml of PBS con-taining 40 mg ml-1 propidium iodide and 10 mg ml-1 RNase Aand incubated at 37°C for 30 min. The DNA content of pro-pidium iodide-stained cells was analysed with a FACSCaliburanalytical flow cytometer (Becton Dickinson, Oxford, UK) witha HeNe laser (excitation wavelength 488 nm). The percent-ages of cells with DNA content < 2C, 2C, 2–4C, 4C and 8Cwere determined using the CellQuest Pro software version6.0 (Becton Dickinson). Gates for the different cell phases/populations were set manually. For each time point, 10 000events were measured.

Lipid extraction and phosphorous determination

Trypanosome lipids were extracted with chloroform : metha-nol (2:1, by volume) as described before (Signorell et al.,2008a). Phospholipids were separated by two-dimensional



TLC using solvent system 1 (chloroform : methanol : ammo-nia : water, 90:74:12:8, by volume) for the first and solventsystem 2 (chlorofom : methanol : acetone : acetic acid :water, 40:15:15:12:8, by volume) for the second dimension(Bütikofer et al., 1989). On each plate, appropriate lipid stan-dards were run alongside the samples to be analysed. Lipidspots were visualized by exposing the Silica Gel 60 plates toiodine vapour. Phospholids were scraped from the plates andlipid phosphorous was determined as described before(Signorell et al., 2008a).

Isolation of mitochondria and ATP production assay

ATP production was measured in mitochondria isolated fromtrypanosomes using 0.01% (w/v; final concentration) digito-nin exactly as described before (Bochud-Allemann andSchneider, 2002). The isolation of mitochondria from unin-duced and induced cells was performed using equal cellnumbers as starting material to assure equivalence betweenthe sample preparations. To determine the amount of non-mitochondrial ATP, samples were treated with 6.7 mg ml-1

atractyloside for 10 min at 4°C before the ATP assay.

Acknowledgements

We would like to thank Andrew Hemphill, Monika Rauch andJennifer Jelk for assistance during parts of the project. Thework was supported by Swiss National Science FoundationGrant 3100A0-116627 to P.B. and The Wellcome Trust andEP Abraham Trust. K.G. is a Wellcome Trust PrincipalResearch Fellow. P.B. thanks T. Rasmus and M. Bütikofer forvaluable input.

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