WCB is a C2 Domain Protein Deﬁning the Plasma Membrane

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http://www.elsevier.de/protisPublished online date 16 October 2007

1

Correspondine-mail gull@pa

& 2007 Elsevdoi:10.1016/j

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Protist, Vol.
ORIGINAL PAPER

WCB is a C2 Domain Protein Defining the PlasmaMembrane — Sub-Pellicular Microtubule Corset ofKinetoplastid Parasites

Andrea Baines, and Keith Gull1

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

Submitted April 30, 2007; Accepted August 18, 2007Monitoring Editor: George B. Witman

WCB is a protein that locates between the inner face of the plasma membrane and the sub-pellicularcorset of microtubules in Trypanosoma brucei. We provide the molecular identity of WCB andbioinformatic analysis suggests that it possesses a C2 domain implicated in membrane/proteininteractions and a highly charged region possessing characteristics of a putative tubulin-bindingdomain. Functional analyses via RNA interference (RNAi) depletion show that WCB is essential for cellmorphogenesis. Depletion results in gross abnormalities in cell shape, mainly at the cytoskeletal/plasma membrane dynamic posterior end of the trypanosome. Failures in cytokinesis and zoidproduction are also evident. Furthermore, electron microscopy reveals that RNAi-induced trypano-somes lose local plasma membrane to microtubule corset integrity.& 2007 Elsevier GmbH. All rights reserved.

Key words: C2 domain; Kinetoplastida; microtubules; plasma membrane; T. brucei; trypanosomes.

Introduction

The shape and form of many protists are definedby a complex internal cytoskeleton lying directlyunderneath the plasma membrane. In trypano-somes this structure takes the form of a sub-pelli-cular corset of microtubules. The sub-pellicularmicrotubules cover the entire inner surface of theplasma membrane necessitating all membranetraffic, to and from the plasma membrane, tooccur at the flagellar pocket (for reviews see Fieldand Carrington 2004; Gull 2003; Landfear andIgnatushchenko 2001). It is only this flagellarpocket region of the plasma membrane that isdevoid of microtubules. The microtubule array iscross-linked together and is present throughout

g author; fax +44 1865 285691th.ox.ac.uk (K. Gull).

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is article as: Baines A; Gull K WCB is a C2 Domain Prot

etoplastid Parasites, Protist (2007), doi:10.1016/j.proti

the full cell cycle with new microtubules beingadded and the array being inherited in a semi-conservative manner by the two daughter cells(Sherwin and Gull 1989a, 1989b). This form of asub-pellicular corset of microtubules is a char-acteristic feature of both free living and parasitickinetoplastid protozoa. Whilst the biochemistryand structural organisation of the sub-pellicularmicrotubules themselves have been studied indetail (Gull et al. 1986; Sasse and Gull 1988;Schneider et al. 1987; Sherwin and Gull 1989a,1989b) we know little about the proteins that mightact either as microtubule—microtubule or micro-tubule—plasma membrane linkers to orchestrateand regulate cytoskeletal events. A few proteinssuch as MARPs, CAP 15 and CAP17 have beenimplicated as sub-pellicular array microtubule

ein Defining the Plasma Membrane — Sub-Pellicular Microtubule

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2 A. Baines and K. Gull

proteins (Hemphill et al. 1991, 1992; Vedrenneet al. 2002).

We have previously characterised a monoclonalantibody WCB-1 (whole cell body-1) whichdefined a protein that was highly phosphorylatedand intimately associated with the sub-pellicularmicrotubule corset (Woods et al. 1992). The WCBprotein was located on the plasma membrane-facing side of the sub-pellicular cage by electronmicroscope immunogold studies (Woods et al.1992). WCB is tightly bound to the detergentinsoluble cytoskeleton but only on the sub-pellicular corset—it is not found on the flagellaror spindle microtubules. We now reveal themolecular identity of the WCB protein, using theoriginal monoclonal antibody to screen an expres-sion library. WCB exhibits both an N-terminalregion C2 domain normally found in proteinswhich interact with membranes (Davletov andSudhof 1993) and a repetitive, charged C-terminalregion which has characteristics of a microtubule-binding domain. We have studied the function ofWCB using inducible RNAi.

Results

The expression library was screened with the well-characterised WCB-1 monoclonal antibody(Woods et al. 1992) and four positive clones werefinally sequenced, which all identified the sameprotein. The resulting sequence was used tosearch the GeneDB Trypanosoma brucei genomedatabase and identified a protein (Tb927.7.3550)with homologs conserved in Trypanosoma cruziand Leishmania major. Synteny of this gene wasconserved between the three kinetoplastids.Homologs were also detected in the otherkinetoplastid protozoa with genome sequence inGeneDB but no convincing homologs weredetected in any other organisms. TheTb927.7.3550 predicted protein is 1241aa inlength with a predicted mass of 138.1 kD and aP.I. of 5.3. We have termed this protein WCB.Bioinformatic analysis of WCB revealed it topossess a C2 domain close to the N-terminus(aa130—210). C2 domains are often Ca2+- depen-dent membrane targeting domains found in manyproteins with a role in signal transduction ormembrane trafficking (Davletov and Sudhof1993). The C-terminus of WCB is dominated bya region of repetitive, charged motifs (Fig. 1A).There are five ‘‘repeats’’ of 32 amino acids builtaround a central KSAED sequence. The repeatsare imperfect and contain many acidic and basic

Please cite this article as: Baines A; Gull K WCB is a C2 Domain Prot

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amino acids — 270 residues out of 580 arecharged in this region, compared to 193 chargedresidues out of 661 in the remaining sequence.The overall PI of this domain is 6.37. Whilst notshowing complete conservation of sequence, thisarea fits a general pattern of basic and acidicresidue repeats seen amongst many other micro-tubule-associated proteins in different organisms(Felgner et al. 1997; Goode et al. 1997; Noble et al.1989). Eleven other C2 domain containing proteinswere identified from GeneDB and alignmentsprepared. No significant homology existedbetween the 12 genes and the C2 domains werevariously located, suggesting that these C2domain proteins do not form a common familybut present as individual proteins with a commonmembrane targeting motif. Finally, bioinformaticanalysis revealed a large number of potentialphosphorylation sites for a number of kinaseswithin the WCB protein.

WCB is expressed in both procyclic and blood-stream cells as judged by Western blotting (datanot shown). The function of WCB was studiedusing an RNAi system in procyclic cells since weknow most about WCB localisation and biochem-istry in these cells (Woods et al. 1992). Non-induced trypanosome populations maintained anormal growth profile over 72 h. However, after24 h the induced population exhibited a slightreduction in growth which became more extremewith time such that between the 48 h and 72 hpassages little growth occurred (Fig. 1B). Mor-phological examination of the induced cell popu-lation showed that at 24 h some cells exhibited arather enlarged and rounded posterior end. After48 h this phenotype was more extreme and cellsexhibited a fattened posterior end. Zoids(non-nucleated cytoplasts with a kinetoplast, 1K0N)were present and clumped cells were visible in theculture. After 72 h very large clumps were obser-vable in flasks by the naked eye. These clumps ofcells were easily dispersed by a few momentsshaking such that individual cells were observedby microscopy. Western blot analysis using theWCB-1 monoclonal antibody along with L8C4 antiPFR monoclonal antibody (as a loading control)(Kohl et al. 1999) showed that RNAi was extremelyeffective at reducing WCB protein expression.There was a large reduction in the level of WCB at24 h and further reduction through the 72 h periodused for phenotype analysis (Fig. 1C).

Counts of kinetoplast (K) and nuclear (N)numbers in the cells provided information onprogression of cell cycle events under depletionof WCB protein. Even at 24 h, major changes had


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A Highly charged repetetive regionC2 DomainB

1 -MSIFGEPLPHEDDTATSLIRREGFPPFSTYDQMPVHYACSCCAEINPSRGRNHLLRTAKQ

121 -SRRQTNTTRLIVTVHHGKSINVDPRDPVSVLVRCGAFEGQTAKVPRGRSAICTWEELFEF181 -PYPNEEEGLEVLVVDDTLPAENDHMFGGIVVPPHALRNRARGDEETLPVCAAGEMHRSYG241 -RMKETPMGSIVVSWYVKRDGEDDADGAALKQNLEGGPINCNFVVHRLFQYTDNGALPYNG301 -GVLCLLRDTDDNCSESGLYTFNSEGAMSSSPYYGKGGCTYLPQDRSQMLQLITPKKLGHV361 -LICVTKDEVENEEDEELIVVGAVPLDFEKLYHKGSAVLLVESKVKEDVLWGEIAVEWGII421 -SYATIQKSNEEDRQGTQMSNDRTGQRDGSGPRESLFLTVVRGLNLTDREGEPLAQGQVSV481 -FACDMEGSTYSAPAVVEADGLAHVITWNQEVRFIEMDGGQAFIDVQVLDDKRIVSSGRFE541 -LLNDSDALTVKMHDPQNETVKRGEILVSYKLIRSTEEDDGESKRELSNEKEEEGSSPRRG601 -ASGRSGRGGSKRQSEMRSDQLSSRRNEDGSEYASDSREKSAEKGRSAREKSPSKQRSQKR661 -SPRSSRKSNSKGSTARQTSRSPKSTSPKRNEEEGGRYLDELDEQQVKSAEDGRSPKERPL721 -SKRSEEERGSDRFNESAAKSAEDDRSAQDKPLSKRNEENEDLEELDEQQVKSAEDDRSAQ781 -ERPLSKRSEEERGSDRFNESAAKSAEDDRSAQDKPLSKRNEEDEDLEELDDPQGHAAEDE841 -QHPSGGERKKKRSPKESEKSRKSPKEGKEKRSPRETTGKSKEESEKSRKSPKEGKEKRSP901 -RETTGKSKEGSEKSRKSPKKSKEKRTPTEAAGKSADEEGYGDDGPLSKRTAEEESLEELD961 -EPQGLAGADERYPVKEDEYTRSPKELAEEDLEELDEPKAEEPTEEQPLSKRNEEGGERLS1021 -SKGSEGRFEKEGGKEQRTVKLDVPPTPSPDRKPPIGKGDERSAKKKAASQGGSEKRSGSG1081 -KKPVSPEAATKKGRLSGQRDSAENVRGRGDSAGRTRAAEGKPSTSEAESSDVGAAANTRN1141 -RSPRSSVRRPSERLDEHNFSISPGASLTSGNWQPWHPTKNASNEDHIPLEKRTTFRNRTE1201 -TIKQLEMRSRDSRLSSGHPSGNRDSSRRASTNRESSVGRGF

61 -EKELEREKLMQINIIEERAKNPHPSIPRPRTSNLQRVMEDEELNQPAIPLPPTTLDELRK

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Figure 1. Bioinformatic and phenotypic analyses of WCB. A: Diagram showing domain positioning and thededuced amino acid sequence of Tb927.7.3550. In the amino acid sequence, the N-terminal C2 domain isunderlined and the repetitive, highly charged C-terminal domain is boxed in grey. B: Three independentgrowth curves of non-induced and induced WCB RNAi cells are shown (labelled non-induced/induced 1,2,3).Decrease in growth rate after induction becomes more pronounced over time. C: Western blot of non-induced and induced WCB RNAi cells showing a marked drop in WCB expression after induction. Anti-PFR2(L8C4) antibody is used as a loading control. D: Kinetoplast (K) and nuclear (N) counts of non-induced andinduced WCB RNAi cells showing a marked increase in abnormal cells after induction. Bars labelled ‘‘Other’’represent multinucleate, multi-kinetoplast cells.

3C2 Domain Protein

occurred to the K/N ratios in the population incontrast to controls (Fig. 1D). There was asignificant drop in the number of 1KIN and 2KINcells (to 22% and 15%, respectively). The 2K2Ncell population had risen to be the most abundantsub-population (28%) with an increase in the


Corset of Kinetoplastid Parasites, Protist (2007), doi:10.1016/j.proti

number of abnormal 1K2N cells and zoids(1K0N). These latter abnormal cell types, togetherwith the large rise in the 2K2N population, stronglysuggested that depletion of WCB delays, inhibitsor induces mistakes in cytokinesis. After 48 hinduction very few normal cells were present.


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The population consisted almost entirely of zoids(1K0N cytoplasts) and multinucleated/multikineto-plast (51% and 38%, respectively) cells (Fig. 1D).This pattern was maintained at 72 h.

In normal and RNAi non-induced trypanosomesthe WCB-1 monoclonal detects WCB across thewhole of the cell body but not the flagellum(Woods et al. 1992) (Fig. 2). On RNAi inductionWCB disappeared from the posterior end of thecell with the immunofluorescence signal often

Phase

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Figure 2. WCB depletion occurs after induction whilstRNAi cells labelled with WCB-1 and TAT-1 (anti-alpha tRNAi cells labelled with WCB-1 and TAT-1 MAbs. WCB-1posterior end of the cell, although TAT-1 labelling showinduced WCB RNAi cells labelled with WCB-1 and L6BWCB RNAi cells labelled with WCB-1 and L6B3 MAbpopulation, flagella and corresponding flagellum attach



being retained at the anterior portion. At 48 h cellsare rather rounded at the posterior end. Some,such as that seen in Fig. 2B, appear to be 1K2Ncells (presumably having lost 1K in a zoid that hascleaved off). Flagella and their associated cyto-plasmic flagellar attachment zone (FAZ) filament,defined by the monoclonal antibody L6B3 (Kohlet al. 1999), continued to be formed even inthe absence of effective new WCB synthesis.Fig. 2D shows a very aberrant multinucleated,

WCB-1 TAT-1

WCB-1 L6B3

other proteins are unaffected. A: Non-induced WCBubulin) MAbs. B: Induced (24 h post-induction) WCB

labelling disappears after induction, initially from thes the microtubule network remains intact. C: Non-3 (anti FAZ) MAbs. D: Induced (48 h post-induction)s. Despite gross abnormalities in the induced cellment zones are still produced. Scale bar ¼ 2mM.


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5C2 Domain Protein

multi-kinetoplastid cell which has the WCB signalstill at what we presume is the original anteriorend. However, even though extremely abnormal, itclearly possesses multiple flagella and multipleFAZ filaments. Staining with the anti a-tubulinTAT-1 monoclonal antibody (Woods et al. 1989)revealed that although WCB is missing initially atthe posterior ends of cells and then moreextensively, the microtubule network seemed tobe present throughout (Fig. 2B).

We next examined the ultrastructural effects ofRNAi-induced WCB protein depletion on thestructure of the trypanosome. Thin section elec-tron micrographs of non-induced trypanosomesrevealed them to have normal ultrastructure with asub-pellicular corset of evenly spaced microtu-bules when viewed in both glancing section andcross-section (Fig. 3A and C). In RNAi-inducedtrypanosomes, the sub-pellicular corset of micro-tubules was present and relatively normal. In thinsection electron micrographs this was best seenin glancing sections where sweeps of the array arevisible (Fig. 3B and D). However, more generalaspects of the plasma membrane—microtubulestructure appeared abnormal. First, cells exhibitedexamples of flagella where the flagellar axonemetogether with a paraflagellar rod (PFR) andflagellar membrane were directly surrounded byclosely associated flagellar pocket membrane(Fig. 3F). Second, induced cells exhibited manyexamples where plasma membrane containingcytoplasm was blebbing from the cell surface.Examples of this are shown in Fig. 3G—K. Itappeared that the normally close connectionbetween the sub-pellicular microtubule corsetand membrane had been locally loosened. Weanalysed the culture supernatant of these inducedcells for the presence of the surface proteinprocyclin by Western blotting, but could detectnone, suggesting that these blebs are not releaseden masse into the medium. Measurements of cellperipheries from a number of micrographs of non-induced and induced cells were carried out andthe average number of blebs per 10mm wascalculated. For non-induced cells, this figure was0.2 (70.14) and for induced cells the figurewas 5.7 (71.1). Micrographs of other mutant celllines generated in our laboratory (Dawe et al.2005; Moreira-Leite et al. 2001) were studied tosee if this blebbing phenomenon was a generalfeature of dying cells with cytokinesis defects, butblebs were not observed in these cases.

In order to assess the general integrity of thesub-pellicular cytoskeleton we viewed cytoskele-tal preparations both by thin section electron



microscopy and by whole mount negative stainelectron microscopy. Thin sections of cytoskele-tons of non-induced trypanosomes revealed thenormal pattern of flagella profiles with cytoskeletalsub-pellicular ‘cages’ often surrounding thenuclear remnant. Glancing sections of the sub-pellicular array of non-induced cells usuallyrevealed the microtubules to be evenly spaced(Fig. 4A, B, D). However, cytoskeletons of RNAi-induced trypanosomes revealed a significant lossof overall integrity. Sub-pellicular corsets ofmicrotubules appeared less well organised afterdetergent treatment and glancing sectionsrevealed rather uneven spacing (Fig. 4C, E).

Finally, we examined whole mount, negativelystained cytoskeletons. Cytoskeletons from non-induced cells showed a normal appearance withexamples of 1K1N and 2K2N cells seen in Fig. 5A,B. Zoids were apparent in the induced populationand zoid cytoskeletons often exhibited a looserorganisation of microtubules at a ‘swollen’ poster-ior end (Fig. 5C), although a more normal micro-tubule structure is visible at the anterior end of thecell, with ordered microtubules running parallel tothe flagellum. Examples of cytoskeletons of multi-flagellated and multinucleated trypanosomes areshown in Fig. 5D, E. Both of these micrographsexhibit the characteristic features of these WCBRNAi-induced cells and cytoskeletons. The sub-pellicular microtubule cytoskeleton in the mainbody of the cell at the swollen posterior end isdisorganised. Under the influence of detergent thisend of the cell appears to have been catastrophi-cally lysed by the preparation procedure with theinter-microtubule organisation being extremelydisorganised. However, even in these cells theanterior end organisation appears much morenormal, with ordered microtubule cytoskeletalarrays running alongside the emerging flagella(arrows in Fig. 5D, E). In Figure 5D, anotherexample of a zoid can be seen (arrowhead). Thiscell is defined as a zoid and not a continuation ofthe large multiflagellated and multinucleated cellbecause the ends of two flagella are clearly seen(white arrows) emerging from the large cell — theflagellum of the zoid cytoplast is not attached tothis cell.

Discussion

WCB was named after the original observation thatthe WCB-1 monoclonal antibody recognized the‘whole cell body’ cytoskeleton of T. brucei but notthe flagellum. Our previous immunolocalisation


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A B

EF

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Figure 3. Thin section TEMs of non-induced and induced (48 h post-induction) WCB RNAi cells, withinduced cells showing ultrastructural abnormalities. A and C: Non-induced cells showing regularly spacedmicrotubules. B and D: Induced cells showing that although microtubules are present and relatively normal,some abnormalities in their alignment and spacing occur. E: Non-induced cell showing a normal flagellum in aflagellum pocket. F: Induced cell with an aberrant flagellum containing a paraflagellar rod structure within aflagellum pocket. G—K: Plasma membrane blebs away, suggesting loss of integrity of the plasma membranefrom the sub-pellicular microtubule corset. Scale bar ¼ 0.2 mM.


Please cite this article as: Baines A; Gull K WCB is a C2 Domain Protein Defining the Plasma Membrane — Sub-Pellicular Microtubule

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7C2 Domain Protein

studies at the electron microscope level (Woodset al. 1992) revealed WCB to be located on theplasma membrane-facing side of the sub-pellicu-

A B

D

C

E



lar microtubule corset. It is tightly associated withthe microtubule cytoskeleton. This location impli-cated WCB in either directly or indirectly linkingthe microtubules to the plasma membrane(Woods et al. 1992). Our present demonstrationof the molecular identity of WCB is intriguing inthis respect. First, the C-terminus of the proteincontains a repetitive region with a high percentageof interspersed basic and acidic residues (K, R, E,D). Tubulin-binding domains in microtubule-asso-ciated proteins are rather diverse but this region isreminiscent of the characterised highly repetitiveKKE motifs in MAP1B and the repetitive KK-containing motifs of Tau in mammalian cells(Goode et al. 1997; Noble et al. 1989). Second,bioinformatic analysis suggests that WCB proteinpossesses a C2 domain near the N-terminus. Thisdomain was originally identified as a conservedCa2+-dependent domain in classical proteinkinase C (PKC) protein and is important in lipidand membrane binding (for review see Cho andStahelin 2006). To date a large number of C2domain containing proteins have been identifiedand they exhibit diverse involvements in signaltransduction or membrane trafficking. Alpha, betaand gamma PKCs have C2 domains that areCa2+-dependent and possess key aspartic acidresidues involved in the Ca2+-binding and regula-tion. However, non-classical PKCs and manyother proteins possess C2 domains that do notbind Ca2+ and in these the key aspartic acidresidues are missing (Cho and Stahelin 2006).WCB appears to fall into this Ca2+-independentgroup of C2 domain proteins. Some of these Ca2+-independent C2 domains have been reported tobind membrane but many of them may also beinvolved in protein—protein interactions. There isrecent evidence that the C2 domain can bindmultiple targets such as multiple lipids or lipids/proteins combinations and can operate in mole-cular scaffolds at the membrane (Cho andStahelin 2006). Future work beyond this studymay resolve whether this WCB C2 domain ismediating plasma membrane interaction directly

Figure 4. Comparison of thin-section electronmicrographs of cytoskeletal preparations of non-induced and induced WCB RNAi cells shows howinduced cells lose overall integrity. A, B and D: Non-induced cells with regularly spaced and orderedmicrotubules. C and E: Induced cells (48 h post-induction) with much looser associations betweenmicrotubules and more random spacing. Scalebar ¼ 0.2 mM.


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A B C

D

E

Figure 5. Whole mount negatively stained cytoskeletons of induced WCB RNAi cells reveal a swollenposterior end with a disorganized microtubule cytoskeleton. A and B: Non-induced trypanosomes at the1K1N and 2K2N stage (respectively) of the cell cycle. C: Zoid with a rounded posterior end, but moreorganised microtubules at the anterior end are produced following induction (72 h post-induction). D andE: Induction (72 h post-induction) also produces multinucleated, multi-kinetoplast cells with disorganisedsub-pellicular microtubules at the swollen posterior ends. In comparison to the posterior end of the cells,microtubules at the anterior ends of the large abnormal cells, where the flagella emerge from the cell body,are much more organised (arrows). The large, abnormal cell in D lies over a zoid (arrow head): the ends of twoflagella emerging from the large cell are clearly visible (white arrows) and do not adjoin the flagellum of thezoid cell. Scale bar ¼ 2mM.


through lipid interaction or via interaction withunknown proteins or a combination of both. Plantcells also exhibit interphase bundles of cytoplas-mic microtubules linked to the inner face of the



plasma membrane. Interestingly, a C2 domainprotein has also been implicated in transientbinding both to microtubules and the plasmamembrane in tobacco cells (Gardiner et al. 2001).


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9C2 Domain Protein

Other than the C2 domain, this 90-kD proteinshows no primary sequence homology to WCBbut has been shown to possess phospholipase Dactivity. In a similar manner WCB may participatedirectly or indirectly in linker functions andpossess signalling or regulatory properties. Thecytoskeletal proteins CAP15 and CAP17 havebeen shown to localise to the sub-pellicularmicrotubule corset of T. brucei but, unlike WCB,mainly to the less dynamic anterior end (Vedrenneet al. 2002). It is unclear whether either of theseproteins link between microtubules or betweenmicrotubules and the membrane. However, Vedr-enne et al. (2002) conjecture that an additional 20amino acid hydrophobic domain on CAP17 mightindicate a membrane interaction.

Our functional analysis reveals that upon RNAi-mediated protein depletion the major effect is thatthe cells have increasing difficulty in completingan effective cytokinesis. This results in a dramaticincrease in 2K2N cells and in the production ofzoids (1K0N) which are indicators of cytokineticmistakes. Examination of these abnormal cellsrevealed that the major aberrations in shape andform occur at the posterior end of the cell. Themultinucleated cells apparently perform manyattempts to cleave by constructing new flagella,new flagellar attachment zone filaments andrelatively coherent anterior poles. However, theseanterior pole initiations of cleavage do notprogress through to the full posterior zone result-ing in large multinucleated monstrous cells withmany individual anterior ends each associatedwith a small group of microtubules, FAZ filamentand flagellum. This phenotype is understandablewhen one appreciates that the major site of newmicrotubule assembly is at the posterior end ofcells where the plus ends are located (Robinson etal. 1995). Thus, it is at this growing posterior endthat the major effects would be seen of depletionof a protein such as WCB that interacts with themicrotubules at the inner face of the plasmamembrane. Although there is a ‘ballooning’ of thisend of the cell in RNAi depletion, microtubulesare still present and are still orientated together.The basic microtubule—microtubule cross-brid-ging appears still to exist. What is almostimpossible to discern by electron microscopy isif there is some rather general dislocation of thecoordination and integration of the membrane—microtubule interaction. The normal microtubulecytoskeleton of trypanosomes involves bothmicrotubule—microtubule connections and micro-tubule—membrane cross bridges. Local disrup-tion of the geometry of the latter could, we



presume, produce concomitant affects on theformer. The phenotype of ‘ballooning’ of theposterior end and blebbing of the cell surfacesuggests a local loss of microtubule corset toplasma membrane-inner face connection in theabsence of adequate WCB. Overexpression stu-dies of the cytoskeletal proteins CAP15 andCAP17 similarly led to the production of zoids,multinucleated cells and failures in cytokinesis(Vedrenne et al. 2002).

One of the major characteristics of the kineto-plastid protozoa, both free-living and pathogenic,is their stable sub-pellicular microtubule corset.This is a case of the extreme biology of trypano-somes. Whilst many other organisms have indivi-dual microtubules that interact with the plasmamembrane and some, such as the land plants andprotists, have groups of sub-plasma membranemicrotubules, few have a complete corset that ispresent throughout the cell cycle. Our initial bio-informatic analysis suggests that WCB is involvedin a trypanosome-specific example of this phe-nomenon. Identification of WCB represents aglimpse into a likely multi-protein macromolecularstructural and regulatory complex (possiblyincluding other proteins such as CAP15, CAP17and MARPs) involved in this corset/inner plasmamembrane face milieu.

Methods

Expression library screening: A lgt11 expression library wasscreened according to standard protocols (Sambrook andRussell 2001) and filters were exposed to a 1/10 dilution ofWCB-1 antibody (Woods et al. 1992) in Tris-buffered saline(TBS)/0.05% Tween-20/1% milk at room temp for 1 h.Following washes in TBS/0.05% Tween-20 the filters wereexposed to 1/20,000 dilution of anti-mouse alkaline phospha-tase-conjugated secondary antibody (Sigma) in TBS/0.05%Tween-20/1% milk at room temperature for 1 h. Followingwashes in TBS/0.05% Tween-20, the filters were exposed to5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazoliumdeveloping solution (BioRad) for approximately 20 min.Positive plaques were identified by dark purple spots on thefilter and were stabbed out from plates. The stab was mashedup in 500ml SM (100 mM NaCl, 10 mM MgSO4 � 7H2O, 50 mMTris-Cl (pH 7.5), 0.01% (w/v) gelatin solution) buffer. Plates ofneat and 10�1 to 10�4 dilutions of this preparation were re-screened. Again, a positive plaque was stabbed out from thecorresponding plate and re-screened. Finally, four positiveplaques were selected, resuspended in SM buffer andamplified by PCR using T3/T7 standard primers. Resultantproducts were sequenced and a GeneDB (http://www.gened-b.org/) search identified a conserved hypothetical protein,Tb927.7.3550.

Constructs and trypanosome transfection: A 581 basepair fragment (from 899—1480bp) of Tb927.7.3550 wasamplified by PCR by specific primers incorporating XhoIand BamHI sites onto the forward and reverse primers,


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respectively (WCB for CCGCTCGAGGTGGTGTGCTGTGTCT-GCTT WCBrev: CGCGGATCCCTGGCGCAGAGTATGTTGAA).The resultant product was cloned into the p2T7-177 inducibleRNAi vector (Wickstead et al. 2002) using the XhoI and BamHIsites. Procyclic T. brucei 29—13 cells (Wirtz et al. (1999)) weretransfected using standard protocols and selected using5 mg ml�1 phleomycin.

Culture and induction of trypanosomes: Cells werecultured in SDM-79 (Brun and Schonenberger 1979) medium,supplemented with 10% fetal calf serum and 5mg ml�1

phleomycin. Prior to induction, phleomycin was removed fromthe culture for three days. For RNAi, cells were induced with1mg ml�1 doxycycline. For growth curves, cells were countedand passaged to a density of 1� 106 cells ml�1 into two flasks;one induced with doxycycline as above, one non-induced.After 24 h, cells were counted again, passaged back to 1� 106

cells ml�1 into new flasks, with or without drug. Counts wererepeated for both flasks at 48 and 72 h post-induction. Countswere repeated on three separate inductions for consistency.

Immunolocalisation studies: For DNA staining, trypano-some cells at zero, 24, 48 and 72 h post-induction of RNAiwere settled onto glass slides and fixed in 4% formaldehydein phosphate buffered saline (PBS). They were stained with1 mg ml�1 4,6-diamide-2-phenylindole (DAPI) and mounted inVectashield (Vector Laboratories).For immunofluorescence, trypanosome cells at zero, 24, 48and 72 h post-induction of RNAi were settled onto glass slidesand were extracted with 1% Igepal CA-630 (Sigma) in PEM(0.1 M PIPES, 2 mM EGTA, 1 mM MgSO4 pH 6.9) for threeminutes before being fixed in 4% formaldehyde in PBS. Cellswere single labelled with WCB-1 (Woods et al. 1992), TAT-1(Woods et al. 1989) and L6B3 (Kohl et al. 1999) and doublelabelled with WCB-1/TAT1 and WCB-1/L6B3. Secondaryantibodies were FITC-conjugated anti-mouse IgG (Sigma),TRITC-conjugated anti-mouse IgG (Sigma) and Rhodamine-conjugated anti-mouse IgM (Chemicon International).Cells were then stained with 1mg ml�1 DAPI and mounted inVectashield. Slides were examined on a Leica Leitz DMRBEmicroscope using a 100�1.4NA oil immersion lens. Imageswere captured on a CCD camera controlled by IPLab software(Universal Imaging) and processed in Adobe Photoshop.

Western blotting: Trypanosome cells at zero, 24, 48 and72 h post-induction of RNAi were washed in PBS-containingprotease inhibitors and were resuspended in a volume ofboiling Laemmli sample buffer to give a concentration of2�105 cells per ml. Samples were run on 10% acrylamide gelsand were then transferred by wet transfer onto nitrocelluloseat 120 mA for 16 h. Western blotting was carried out accordingto standard protocols (Sambrook and Russell 2001) using a1/10 dilution of WCB-1 antibody (Woods et al. 1992) with a1/200 dilution of anti-PFR2 antibody L8C4 (Kohl et al. 1999), asa loading control, in TBS/0.05% tween/1% milk. Secondaryantibody was peroxidase-conjugated anti-mouse IgG (Sigma),diluted 1:20,000 as before. Membranes were covered withWestern Lightning Chemiluminescence Reagent Plus (Perki-nElmer), exposed to Kodak X-OMAT LS film and developed in aKodak X-OMAT 2000 developer.

Preparation of cells for thin-section TEM: Trypanosomecells were fixed at zero, 24, 48 and 72 h post-induction ofRNAi in 2.5% glutaraldehyde in tissue culture medium.Cytoskeletons were generated by washing the cells (at 0,24, 48 and 72 h post-induction of RNAi) with 1% Igepal-CA630(Sigma) in PEM for five minutes. The cells were pelleted andfixed in 2.5% glutaraldehyde, 2% paraformaldehyde and0.1% picric acid in 100 mM phosphate (pH 6.5) for 2 h at 41Cfollowed by 1% osmium tetroxide in 100 mM phosphate buffer(pH 6.5) for 1 h at 41C. In both cases, the fixed material was



stained en bloc with 2% aqueous uranyl acetate for 2 h at 41C.The material was dehydrated through a graded series ofacetone and propylene oxide and was then embedded inEpon resin for sectioning.

Acknowledgements

This work was supported by grants from theWellcome Trust and EP Abraham Trust. KG is aWellcome Trust Principal Research Fellow. Wethank past and present members of our group fordirect support of this work and for generousdiscussions, Mike Shaw for assistance with theEM and Christine Clayton for the generous gift ofthe expression library.

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WCB is a C2 Domain Protein Deﬁning the Plasma Membrane