flagellum elongation is required for correct structure ... · organisation, orientation and...

3704 Research Article

IntroductionAfrican trypanosomes are protozoan parasites responsible forhuman tropical diseases, such as sleeping sickness. This neglecteddisease is fatal in the absence of treatment. No vaccine is availableand current therapy relies on the use of toxic drugs, including arseniccomponents that lead to patient death in 2-10% of cases.Trypanosomes proliferate in the bloodstream of their host asextracellular parasites and escape the immune response by using asophisticated process of antigenic variation. The surface of thetrypanosome is covered with a dense coat made of a single type ofprotein (variant surface glycoprotein or VSG) that is periodicallyreplaced. The genome contains ~1000 different VSG genes andpseudogenes that can undergo recombination events to ensure rapidmodification of this repertoire (Berriman et al., 2005).

Endocytosis and exocytosis exclusively take place at the flagellarpocket, a surface microdomain found at the posterior end of thecell. This restriction is due to the presence of a dense network ofsubpellicular microtubules, which prevents fusion-fission events atall other surface locations (Gull, 1999). The flagellar pocket is adepression of the plasma membrane from which the single flagellumemerges and represents barely 1% of the total cell surface (Overathand Engstler, 2004). Freeze-fracture studies demonstrated thatmembranes of the flagellar pocket and of the flagellum itself are

closely opposed at the neck of the flagellar pocket (Yoshikawa etal., 1992). Nevertheless, antibodies that bind to the VSG coat arerapidly cleared by efficient endocytosis of VSG and boundantibodies, followed by recycling of the VSG proteins after antibodydissociation in endosomes (Engstler et al., 2004). The forwardmotility of the parasite in the bloodstream ensures efficient transferof bound antibodies from over the cell surface to the posteriorflagellar pocket via hydrodynamic flow (Engstler et al., 2007).

Little is known about flagellar pocket molecular identity andabout its formation during the cell cycle. The new flagellum isconstructed in the existing flagellar pocket, next to the matureflagellum (Absalon et al., 2008; Briggs et al., 2004; Davidge et al.,2006; Grasse, 1961). Although initial elongation of the newflagellum occurs within the same flagellar pocket, basal bodiesmigrate apart (Robinson et al., 1995) and two separate flagellarpockets are visible, each one with a single flagellum. A definedcytoskeletal structure called the flagellar pocket collar (FPC) isvisible at the neck of the flagellar pocket and remains associatedwith the flagellum upon detergent extraction (Sherwin and Gull,1989). A molecular component of the collar, termed BILBO1, hasbeen identified recently and has been shown to be essential for bothcollar and flagellar pocket formation, but not for basal bodyduplication and flagellum growth (Bonhivers et al., 2008b). Absence

In trypanosomes, the flagellum is rooted in the flagellar pocket,a surface micro-domain that is the sole site for endocytosis andexocytosis. By analysis of anterograde or retrogradeintraflagellar transport in IFT88RNAi or IFT140RNAi mutantcells, we show that elongation of the new flagellum is notrequired for flagellar pocket formation but is essential for itsorganisation, orientation and function. Transmission electronmicroscopy revealed that the flagellar pocket exhibited amodified shape (smaller, distorted and/or deeper) in cells withabnormally short or no flagella. Scanning electron microscopyanalysis of intact and detergent-extracted cells demonstratedthat the orientation of the flagellar pocket collar was morevariable in trypanosomes with short flagella. The structuralprotein BILBO1 was present but its localisation and abundancewas altered. The membrane flagellar pocket protein CRAM

leaked out of the pocket and reached the short flagella. CRAMalso accumulated in intracellular compartments, indicatingdefects in routing of resident flagellar pocket proteins.Perturbations of vesicular trafficking were obvious; vesicleswere observed in the lumen of the flagellar pocket or in theshort flagella, and fluid-phase endocytosis was drasticallydiminished in non-flagellated cells. We propose a model toexplain the role of flagellum elongation in correct flagellarpocket organisation and function.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/121/22/3704/DC1

Key words: Flagellum, Flagellar pocket, Endocytosis, Intraflagellartransport

Summary

Flagellum elongation is required for correct structure,orientation and function of the flagellar pocket inTrypanosoma bruceiSabrina Absalon1,2,*, Thierry Blisnick1,*, Mélanie Bonhivers3, Linda Kohl4, Nadège Cayet5,Géraldine Toutirais2, Johanna Buisson1, Derrick Robinson3 and Philippe Bastin1,2,‡

1Trypanosome Cell Biology Unit, Pasteur Institute and CNRS, 25 rue du Docteur Roux, 75015 Paris, France2Dynamique et Régulation des Génomes, Muséum National d’Histoire Naturelle, INSERM and CNRS, 43 rue Cuvier, 75005 Paris, France3Microbiologie Cellulaire et Moléculaire et Pathogénicité, Université Bordeaux 2 and CNRS, 146 Rue Léo Saignat, 33076 Bordeaux, France4Biologie Fonctionnelle des Protozoaires, Muséum National d’Histoire Naturelle, 61 rue Buffon, 75005 Paris, France5Plateforme de Microscopie Electronique, Pasteur Institute, 25 rue du Docteur Roux, 75015 Paris, France*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

Accepted 14 August 2008Journal of Cell Science 121, 3704-3716 Published by The Company of Biologists 2008doi:10.1242/jcs.035626

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3705Flagellum and flagellar pocket formation

of a flagellar pocket results in multiple cellular defects and is lethalin both procyclic and bloodstream trypanosomes.

These data raise the question of the coordination of these criticalevents of the trypanosome cell cycle: basal body duplication,flagellum construction and flagellar pocket formation. At least threehypotheses can be proposed: (1) the FPC duplicates and the newflagellum is then introduced in the new FPC; (2) the emergence ofthe new flagellum triggers the duplication of the FPC; or (3) bothevents take place and are coincidental. The availability of RNAimutants, in which formation of the new flagellum can be halted(Absalon et al., 2008; Absalon et al., 2007; Davidge et al., 2006;Kohl et al., 2003), opens up the ability to evaluate the role of theflagellum in flagellar pocket duplication. We used procyclictrypanosome cell lines where expression of genes required forintraflagellar transport (IFT) had been silenced. IFT is a dynamicprocess that is required to construct flagella (Cole et al., 1998;Kozminski et al., 1993) and is conserved in almost all flagellatedeukaryotes, from protists to humans (Kohl and Bastin, 2005). Ouranalysis demonstrates that when the new flagellum is too short orabsent, a flagellar pocket is still made, showing that its formationis independent of flagellum growth. However, its structure, itsorientation in the cell body and its functions are modified. Wepropose a model explaining the different steps of flagellum andflagellar pocket formation in trypanosomes.

ResultsBasal body duplication and segregation take place in theabsence of flagellaAs basal body duplication plays a major role in the trypanosomecell cycle (Robinson and Gull, 1991), it is important to first evaluatewhether the basal body duplicates in the absence of a flagellum.Uniflagellated trypanosomes possess a mature basal body that iscontinuous with the flagellum and a shorter immature pro-basal body(Sherwin and Gull, 1989). Two sets of IFT mutants created by RNAiwere used for this analysis. IFT88 and IFT172 (members of IFTcomplex B) are required for anterograde transport (from base totip) and their silencing blocks flagellum assembly (Absalon et al.,2008; Kohl et al., 2003). IFT140 (IFT complex A) and DHC1bgenes encode proteins that are essential for retrograde IFT (transportof material from tip to base of the flagellum). Their silencing initiallyleads to the formation of a short dilated flagellum that is filled withIFT particles as a result of a failure in recycling IFT material(Absalon et al., 2008). This results in a situation where cells cannotbuild a new flagellum or where a tiny abnormal new flagellum isformed but not elongated. In both cases, membrane extension stilltakes place, producing a flagellar ‘sleeve’, which does not containcytoskeletal elements (Absalon et al., 2008; Davidge et al., 2006).

Two markers were used to examine basal body duplication. Themonoclonal antibody mAb22 detects an as yet unidentified antigenlocalised at the region of the mature basal body and the pro-basalbody within a structure called the tripartite attachment complex,which links the kinetoplast to the basal body (Bonhivers et al.,2008a). We also used a rabbit antiserum recognising thetrypanosome basal body component protein (TBBC), a 164 kDacoiled-coil-rich protein (Dilbeck et al., 1999). Previous data indicatethat the anti-TBBC antibody labels only one spot (Absalon et al.,2007; Dilbeck et al., 1999), suggesting that it could be a differentialmarker of the mature basal body or of the pro-basal body. Controlcells were labelled with mAb22, anti-TBBC and DAPI to revealthe nucleus and the kinetoplast, which is always linked to the basalbody (Robinson and Gull, 1991), thus providing a marker of basal

body position. Cells with one flagellum exhibited two spots formAb22, one corresponding to the fibres of the mature basal body(continuous with the flagellum, which is clearly visible by phasecontrast imaging) and one corresponding to the fibres of theimmature pro-basal body (Fig. 1Aa). Both spots were found in closeproximity to the kinetoplast, in agreement with mAb22 localisationat the proximal region (Absalon et al., 2007; Bonhivers et al., 2008a).By contrast, the same cells displayed only a single spot for TBBC,localised at the distal region of the basal body and in continuitywith the axis of the flagellum (Fig. 1Aa). When cells duplicatedtheir basal bodies, four spots were stained with mAb22 and onlytwo with the anti-TBBC antiserum (Fig. 1Ab). This pattern washighly reproducible and demonstrates that TBBC can be used as amarker of the older basal body that is associated with the flagellum.

Double staining experiments were performed on cell lines whereexpression of genes encoding either a complex A protein (IFT140)(Fig. 1B,C) or a complex B protein (IFT88 or IFT172)(supplementary material Fig. S1) had been silenced by RNAi.Inhibition of IFT blocks formation of the new flagellum but doesnot affect the existing flagellum (Absalon et al., 2008; Absalon etal., 2007; Davidge et al., 2006; Kohl et al., 2003). When formationof the new flagellum was inhibited, two basal body complexes couldbe detected: one associated with the old flagellum and one close tothe posterior kinetoplast (Fig. 1B). Both displayed two spots formAb22 and one for TBBC, exactly as in control cells, demonstratingthat replication of the basal body took place normally (Fig. 1B).Once such a cell divides, it produces two daughters: one with aflagellum and one without a flagellum. Basal body duplication goeson in cells without a flagellum, as shown by the presence of a secondbasal body complex, stained with mAb22 and the anti-TBCC (Fig.1Ca-a�). These cells fail to divide but can complete S phase andundergo mitosis, thus becoming multinucleated (Kohl et al., 2003).They are also able to duplicate their basal bodies and acquire TBBCnormally, as shown by the presence of two spots for mAb22 alwaysassociated with a single spot for TBBC (Fig. 1Cb-b�). However,basal body migration is severely limited, as previously reported(Absalon et al., 2007).

Electron microscopy analysis of induced IFT RNAi cellsconfirmed the presence of multiple basal bodies with tripletmicrotubules (at the proximal end) and typical extensions (Fig. 1D).However, the transition region of basal bodies appears shorter inthe absence of a flagellum (203±78 nm in IFT88RNAi cells comparedwith 377±37 nm in wild-type controls, n=18), suggesting thatelongation of the new basal body is not complete. In conclusion,IFT is only required for flagellum construction and possibly basalbody elongation, but not for basal body duplication and acquisitionof the TBBC protein.

Modification of flagellar pocket shape and contentAs basal bodies duplicate, we asked whether a flagellar pocket couldbe formed in these conditions. The presence and structure of theflagellar pocket was investigated by transmission electronmicroscopy (Fig. 2). In wild-type cells, the basal body of theflagellum is found underneath the base of the flagellar pocket, withits proximal part made of triplet microtubules found within the cellbody (Fig. 2A). The transition region is made of nine doublets ofmicrotubules surrounded by the flagellar membrane and is foundin the lumen of the flagellar pocket. When the flagellum exits theflagellar pocket, it now contains a complete axoneme made of ninedoublets of microtubules with dynein arms and radial spokes, anda central pair of microtubules. A large extra-axonemal structure

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called the PFR is only assembled after the flagellum exits theflagellar pocket (Bastin et al., 1996). The flagellum membrane isin close contact with that of the collar of the flagellar pocket andspecific cytoskeletal structures made of electron-dense material arevisible (Fig. 2A, arrows). On electron micrographs, the flagellarpocket exhibits a typical flask shape and the flagellum is positionedasymmetrically, defining a larger and a thinner lumen side foundat the anterior and posterior ends, respectively. Vesiclescorresponding to endosomes or exosomes are frequently seen inthe cytoplasm in close proximity to the flagellar pocket.

The ultrastructure of the flagellar pocket was analysed inIFT88RNAi, IFT172RNAi, IFT140RNAi and DHC1bRNAi cells atinduction times where assembly of the new flagellum was inhibited(Fig. 2B-E). The flagellar pocket was visible in all induced samplesbut its shape was considerably modified, with variable profiles. Cellsthat possessed only a short basal body often presented an abnormallysmall, but recognisable flagellar pocket, without a clear asymmetry(Fig. 2B,C). The structure of the flagellar pocket was alsoinvestigated in IFT140RNAi and DHC1bRNAi mutant cells that stillpossessed a short flagellum where microtubules elongated beyondthe basal body and often presented excessive amounts of IFT-likematerial (Fig. 2D, arrowheads). These exhibited flagellar pocket ofvarious shapes: either deeper than usual (Fig. 2D) or distorted (Fig.2E).

Vesicular material was often present in the lumen of the flagellarpocket (Fig. 2D-E), a feature rarely observed in procyclic wild-typetrypanosomes. Moreover, some vesicles were detected within theshort flagella of retrograde transport mutants (Fig. 2D, see below).Quantitative analysis performed on a total of 146 images from allfour induced mutants revealed that 50-80% of the flagellar pocketsections presented an abnormal profile (Fig. 2G). These values

Journal of Cell Science 121 (22)

correspond to the actual percentage of non-flagellated cells in theculture at the induction time where the analysis was performed (datanot shown). Accordingly, the lowest number of aberrant flagellarpockets was found in the IFT140RNAi induced sample, in agreementwith the lower proportion of non-flagellated cells in this population(50% compared with 75-80% for the other cell lines).

These multiple structural defects could be directly linked tofailure in flagellum elongation but could also result from themorphogenetic defects observed in non-flagellated cells (Kohl etal., 2003). We therefore examined the status of the flagellar pocketin PF16RNAi and in PF20RNAi mutants where the expression of centralpair proteins is inhibited, leading to cell paralysis. These mutantsshare the same morphogenetic defects (reduced basal bodymigration, short cell body, loss of polarity, poor elongation of theFAZ filament) but assemble a flagellum of normal length (Absalonet al., 2007). Transmission electron microscopy revealed that theflagellar pocket of these two mutants was undistinguishable fromwild-type cells (Fig. 2F and data not shown), supporting a directrole of flagellum elongation in flagellar pocket formation. Thesedata indicate that the flagellar pocket is formed, but in the absenceof correct flagellum elongation, it cannot adopt its normal shapeand length.

The orientation of the flagellar pocket is altered in the absenceof flagellum elongationThe above data do not give a global view of the structure andpositioning of the flagellar pocket. We extracted trypanosomes withdetergent to remove the plasma membrane and examined theresulting cytoskeletons by scanning electron microscopy. Strippingthe membrane of wild-type cells exposed the subpellicularmicrotubules with their typical helical pattern, the flagellum and

Fig. 1. Basal body replication takesplace in the absence of a flagellum.(A-C) Merged images of doubleimmunofluorescence staining withmAb22 (marker of the proximal endof the mature and the pro-basal body,green) and the anti-TBBC antibody(marker of the mature basal body, red)of detergent-extracted cytoskeletonfrom non-induced (A, –TET) or 48-hour-induced IFT140RNAi (B, +TET)cells stained with DAPI (blue). Insetsshow magnification of the basal bodyregion. BB, basal body. (C) Non-flagellated cells can duplicate theirbasal body (a) but fail to divide,hence becoming multinucleated cells(b) that accumulate several pairs ofbasal bodies. a� and b� showmagnification of the boxed basalbody regions in a and b.(D) Transmission electron micrographof a section through an IFT172RNAi

cell induced for 72 hours, which hasfour basal bodies (arrows). TET,tetracycline.

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the flagellum attachment zone filament (FAZ) (Fig. 3A). Thistreatment revealed the FPC, which appeared as a horseshoe structurearound the flagellum (Fig. 3A). However, it could also be that theFPC is a ring with a portion hidden by the flagellum. In any case,the posterior part of this structure always appeared to be lifted ontop of the flagellum and in a perpendicular orientation relative tothe main axis of the cell (Fig. 3A). However, this localisation couldbe a consequence of the detergent treatment. We therefore examineduntreated cells using scanning electron microscopy; strikingly, theshape of the FPC could be recognised underneath the plasmamembrane at the point of emergence of the flagellum from theflagellar pocket (Fig. 3B). Its posterior part appeared as an archpositioned above the flagellum at its point of emergence from theflagellar pocket and was found in an orientation very similar to thatobserved in detergent-extracted samples. These data demonstratethe defined orientation of the FPC and validates the detergenttreatment methodology for investigation.

Analysis of cells from the IFT140RNAi mutant with an abnormallyshort flagellum revealed the presence of a FPC structure aroundthe short flagella (Fig. 3Ca-c). Two main differences were noted

compared with controls: (1) the shape of the FPC was more irregularand its thickness was frequently reduced, and (2) its orientationrelative to the main axis of the cell was extremely variable (Fig.3Ca-c). The defect in FPC orientation was also visible on non-extracted cells (Fig. 3D), demonstrating that it was not due todetergent treatment. Non-flagellated cells could not be analysed bythese methods because it was not possible to identify unambiguouslythe FPC in the absence of a flagellum (not shown).

The mis-orientation of the FPC in cells with a short flagellumcould be a secondary consequence of the loss in cell polarity typicalof non-flagellated cells (Kohl et al., 2003). Alternatively, it couldbe incorrectly positioned as soon as the new basal body is unableto assemble a flagellum, for example, as a result of poor basal bodymigration (Absalon et al., 2007). To evaluate the specificity of thismis-orientation phenotype, we analysed the positioning of the FPCat early stages of RNAi knockdown in intact cells or in detergent-extracted cytoskeletons (Fig. 4; supplementary material Fig. S2).In control cells (wild-type or non-induced samples), as the new basalbody is duplicated, the new flagellum is assembled in closeproximity to the existing flagellum. An FPC is seen at the base of

Fig. 2. The flagellar pocket exhibits a modified shape and accumulates vesicles in IFTRNAi mutant cells with a short or no flagellum. (A-F) Sections through theflagellar pocket region of wild-type (WT) trypanosomes (A), of the indicated IFTRNAi mutant cell lines (B-E) or of PF20RNAi cells (F) that were induced for 72hours. The typical flask-shape of the flagellar pocket is altered and vesicles frequently accumulate in the lumen (D,E). A, anterior side of the flagellar pocket; P,posterior side; K, kinetoplast; BB, basal body; TR, transition region. Black arrows indicate the tight contact between the top of the flagellar pocket and theflagellum, the white arrow indicates a vesicle in the flagellar compartment and the arrowheads indicate excessive IFT material typical of retrograde transportdefects. (G) Quantification of sections with the indicated phenotypes in wild-type (WT) and various IFTRNAi cell lines. WT, n=73; DHC1bRNAi, n=51; IFT140RNAi,n=36; IFT88RNAi, n=28; IFT172RNAi, n=31.

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each flagellum (Fig. 4A; supplementary material Fig. S2A),indicating that this structure duplicates rapidly upon formation ofthe new flagellum. In all the cells examined (n>100), the orientationof the FPC associated with the new flagellum was parallel to thatof the FPC of the old flagellum and perpendicular to the main cellaxis (Fig. 4A; supplementary material Fig. S2A). Next, we analysedcells at early stages of IFT protein deprivation (between 48 and 72hours after tetracycline addition) that still possessed an old flagellumbut failed to assemble a normal-length new flagellum. In contrastto control cells, the defined relative orientation of old and new FPCwas already lost in 33% of IFT140RNAi mutant cells (n=30) at thisstage (supplementary material Fig. S2B). These values rose to 67%in IFT88RNAi mutant cells possessing an old flagellum of normallength and a short new flagellum (n=15). This higher proportioncould be related to the fact that IFT88RNAi cells produce extremelyshort flagella compared with IFT140RNAi cells, which often fail toemerge from the flagellar pocket (Absalon et al., 2008). Similardefects in FPC positioning were observed in extracted(supplementary material Fig. S2B) and in intact cells (Fig. 4B).Previous work demonstrated that cells with a normal length old


flagellum but with an abnormally short new flagellum exhibit anormal cytoskeleton (Kohl et al., 2003). Therefore, these resultsreveal that the defined orientation of the FPC is already lost beforethe emergence of the morphogenetic defects at the cellular leveland is more likely to be related to failure in elongating the newflagellum.

The flagellar pocket structural protein BILBO1 shows modifiedlocalisation in non-flagellated cellsWe next examined whether the localisation of a molecularcomponent was also affected. BILBO1 is a structural protein of theFPC that is essential for its formation (Bonhivers et al., 2008b). Incontrol cells possessing a single flagellum, an antibody againstBILBO1 identified a region found precisely around the point wherethe flagellum exits the main cell body, corresponding to the FPC(Fig. 5A, middle cell). Soon after exit of the new flagellum fromthe flagellar pocket, a second region of labelling was detected uponelongation of the new flagellum (Fig. 5A, left cell). Kinetoplast andbasal body segregation then occurs and two signals were clearlyvisible, each associated with a flagellum (Fig. 5A, right cell).

Fig. 3. The defined orientation of theflagellar pocket collar is randomised inIFTRNAi mutants. (A) Scanning electronmicrograph of a wild-type cellextracted with cold Triton X-100. Thewhite rectangle indicates the positionof the magnified area shown on theright. The FPC and the FAZ filamentare indicated by white arrows andarrowheads, respectively. (B) An intactwild-type cell examined by scanningelectron microscopy. The magnifiedarea shows the region where theflagellum emerges from the cell body.The shape of the FPC is visibleunderneath the membrane (whitearrows). (C) Images of IFT140RNAi

cells (a-c) induced for 72 hours andtreated with cold Triton X-100 as in A.The FPC associated with the shortflagellum appears less elaborate and itsorientation relative to the main axis ofthe cell is variable. (D) A non-extractedIFT140RNAi cell after induction ofsilencing for 72 hours. The shape ofthe FPC is clearly recognisable but itsorientation is not perpendicular to themain axis of the cell.

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Knocking down IFT gene expression first led to the formation ofshorter flagella (arrowheads), which possess an apparently normalamount and arrangement of the BILBO protein at the expectedflagellar pocket position (Fig. 5B). However, once flagella werenot visible, the BILBO1 signal was less defined and sometimes lessintense (Fig. 5C). These data were confirmed upon double stainingwith the anti-axoneme marker mAb25 (supplementary material Fig.S2) and indicate that BILBO1 is still synthesised in the absence ofa flagellum and localises to the expected flagellar pocket position;however, its localisation seems modified, a feature that could berelated to the thinner aspect of the FPC in electron microscopy (Fig.3).

The flagellar pocket membrane protein CRAM is affected byincorrect flagellum elongationWe next asked whether membrane flagellar pocket proteins couldstill be routed properly in cells with a modified flagellar pocket.CRAM (cysteine-rich acidic membrane) protein is a flagellarpocket transmembrane protein that is predominantly expressed inprocyclic trypanosomes (Lee et al., 1990). Induced and non-induced IFTRNAi cells were analysed by IFA with anti-CRAMantibodies following paraformaldehyde fixation andpermeabilisation with detergent (Fig. 6A-C). In controltrypanosomes, the presence of CRAM was limited to the posteriorregion where the flagellar pocket is localised (Fig. 6A). By contrast,

Fig. 4. The FPC is disorientated as soon as cells fail toassemble a normal flagellum. (A-B) Scanning electronmicroscopy of untreated non-induced (A) and 48-hour-induced IFT140RNAi cells (B). In controls (A), thetwo flagella emerge from separate FPCs that areparallel. In cells that possess an old flagellum and anaberrantly short new flagellum (B), the orientation ofthe FPC associated with the new flagellum is not fixedrelative to the long axis of the cell nor to theorientation of the FPC of the old flagellum. Arrowsshow the FPC, and arrowheads indicate the flagellarsleeve present in IFT mutants.

Fig. 5. Organisation of the FPCprotein BILBO1 is modified inIFTRNAi cells. (A-C) Immunofluorescence stainingof the indicated cell lines with anantibody recognising BILBO1(green), stained with DAPI(blue). Cells were either non-induced (–TET) or induced for 72hours (+TET). BILBO1 stainingis less defined in cells that lackflagella (C). Arrows indicate cellswith modified BILBO1 stainingand arrowheads indicate shortflagella.

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the CRAM signal was not restricted to the flagellar pocket area inboth IFT88RNAi and IFT140RNAi induced cells (Fig. 6B,C). AsCRAM is a transmembrane protein, this localisation couldcorrespond to material present at the surface of the cell or/and withinvesicles in the cytoplasm. To solve the issue, induced and non-induced cells were fixed and stained with the anti-CRAM antibodieswithout permeabilisation. Surface-bound antibodies were visualisedwith Protein AG coupled to gold particles, and samples wereanalysed by scanning electron microscopy (Fig. 6D-G). Control cellsfailed to reveal significant staining either on the cell body or on theflagellum surface (Fig. 6D), as expected from the restrictedlocalisation of CRAM within the flagellar pocket (Yang et al., 2000),which is not accessible without detergent permeabilisation. Silencingof IFT140 produces cells with a short dilated flagellum, withexcessive IFT material, and other cells without a flagellum (Absalon


et al., 2008). Remarkably, in induced IFT140RNAi cells, the shortdilated flagella exhibited strong CRAM staining at their surface:on average 18 gold particles per μm2 of flagellar surface (Fig. 6E-G), which corresponds to a density ~30-fold higher than in normalflagella of non-induced samples (0.6 gold particles per μm2 offlagellar surface) (Fig. 6D). The old flagellum of induced IFT140RNAi

cells maintained a low level of gold particles similarly to wild-typecells, indicating that CRAM localisation was only modified in thenew flagellum (not shown). Moreover, the ‘flagellar sleeve’ wasalso stained with the anti-CRAM antibody in cells with (Fig. 6E)or without (Fig. 6F) a flagellum. The frequency of gold particleson the rest of the cell surface remained very low, exactly as in controlcells (Fig. 6D-F), revealing that the abundant staining detected inpermeabilised cells by immunofluorescence (Fig. 6B,C) actuallycorresponds to CRAM protein present in intracellular compartments.

Fig. 6. The flagellar pocket proteinCRAM is mislocalised in IFT140RNAi

cells. (A-C) Cells from the indicatedcell lines were fixed andpermeabilised with Nonidet beforeimmunofluorescence with the anti-CRAM antiserum and stained withDAPI. Bottom panels show anti-CRAM signal only (white) and toppanels show merged phase contrast,anti-CRAM (green) and DAPI (blue)images. (D-G) Cells from non-induced (D) and induced (E-G)IFT140RNAi cells were fixed andincubated with the anti-CRAMantiserum without permeabilisation,followed by incubation with ProteinAG coupled to gold particles. Arrowsindicate short dilated flagella typicalof retrograde transport inhibition;arrowheads indicate flagellar sleeves.Scale bars: 1 μm.

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In conclusion, a flagellar pocket membrane protein reaches theflagellum membrane in IFT140RNAi cells and is found in thecytoplasm, revealing defects in the routing of flagellar pocketproteins.

Perturbation of vesicular traffickingThe accumulation of CRAM in cytoplasmic compartments, as wellas its presence on the membrane of short flagella indicates defectsin surface protein trafficking. Furthermore, the presence of numerousvesicles in the lumen of the flagellar pocket (Fig. 2D-F) or in theshort flagella (Fig. 2D) of IFTRNAi cells indicates possible defectsin vesicular trafficking. We therefore examined by transmissionelectron microscopy the presence, nature and distribution ofcytoplasmic vesicles around the flagellar pocket area in DHC1bRNAi,

IFT140RNAi and IFT88RNAi induced cells and compared them withthat in wild-type cells (Fig. 7).

Sections through the flagellar pocket region of wild-typetrypanosomes displayed the usual organelles, includingendosomes and exosomes, the Golgi complex, the flagellum orthe kinetoplast (Fig. 7A-C). We noticed the frequent presence ofa long and narrow cisternal compartment adjacent to the flagellarpocket membrane (Fig. 7B-C, white arrows), which was regularlyfound in flagellar pocket sections (60%, n=72). This compartmentwas more frequently seen at the anterior face of the flagellarpocket, close to the Golgi, and we named it the ‘flatcompartment’. Although its nature and function are still unknown,it could represent a morphological marker of the flagellar pocketenvironment.

Fig. 7. Various vesicles are present inshort flagella and in the flagellarpocket area of IFTRNAi cell lines. (A-C) Wild-type cells. FP, flagellarpocket; BB, basal body; E, endosomes,G, Golgi complex, K, kinetoplast. Thewhite arrow indicates the flatcompartment visible close to theflagellar pocket membrane.(D) Section through the flagellarpocket of an IFT140RNAi cell inducedfor 3 days. Notice the accumulation ofIFT-like material in the short flagellum(IFT excess), the flagellum attachmentzone (FAZ) and the presence of‘double’ (black arrowheads) and bent(black arrow) vesicles close to theflagellar pocket/FAZ region.(E,F) Sections through the flagellum ofDHC1bRNAi cells induced for 72 hours.Notice the presence of round or flatvesicles in the flagellar compartment(some are indicated by short whitearrows). (G,H) IFT88RNAi cells inducedfor 72 hours showing the flagellarpocket region where vesicles are alsoseen in a flagellar sleeve compartment(G) and the Golgi complex, close towhich some double membrane (blackarrowheads) or bent (black arrows)vesicles are frequently identified (H).(I) Quantification of sections that showa flat compartment close to theflagellar pocket membrane (blackbars), at least one double membranevesicle (‘double’, light grey) or at leastone ‘cup-like’ vesicle (dark grey) inthe indicated cell lines. WT, n=63;DHC1bRNAi, n=42; IFT140RNAi, n=36;IFT88RNAi, n=23.

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Numerous large and small vesicles are present in the vicinity ofthe flagellar pocket of the IFTRNAi mutants. Furthermore, someunusual vesicles were observed close to the flagellar pocket andthe Golgi complex. Some vesicles appeared to have two membranes(Fig. 7D,G,H, arrowheads) and others looked bent (Fig. 7D,G,H,arrows). Quantification was performed on a total of 150 sectionsfrom induced IFT88RNAi, IFT140RNAi and DHC1bRNAi cells. Thesevesicles were encountered in almost 30% of the sections inanterograde and retrograde transport mutants (Fig. 7I). This is quiteclose to the frequency of flagellar pocket with vesicles in their lumen(27%, n=146 for four different IFTRNAi mutants) (Fig. 2G).

As noted above, vesicles were found within short flagella in theIFT140RNAi and the DHC1bRNAi mutants that contained IFT-likematerial (Fig. 2D; Fig. 7E,F). In IFT88RNAi and IFT172RNAi cells,a few sections (7/51) showed vesicles in the lumen of the flagellarsleeve albeit without IFT-like material (Fig. 7G). Some, but not all,of these small vesicles exhibited a disk-like shape and werereminiscent of exocytic vesicles (Grunfelder et al., 2003).

As the Golgi complex and the basal body are linked (He et al.,2004), Golgi location was analysed in non-flagellated cells. It wasfound close to the flagellar pocket with the same frequency inDHC1bRNAi, IFT88RNAi and IFT172RNAi (50-60% sections, n=28-51)cells as in wild-type cells (64%, n=72), showing that a flagellumis not required for Golgi association to the flagellar pocket or basalbody region. The flat compartment identified above (Fig. 7A-C)was equally present in the vicinity of the modified flagellar pocketin IFTRNAi cells (Fig. 7I).

Organelle organisation in non-flagellated cellsWe next examined the status of several organelles associated withthe endocytic network using specific markers. First, the p67 proteinwas used as a marker of lysosomes that localise between thekinetoplast and the nucleus (Alexander et al., 2002). This pattern


was reproduced in control non-induced cells (supplementarymaterial Fig. S3A) but was altered in non-flagellated IFT140RNAi

or IFT88RNAi cells where p67 was found in multiple foci at variouslocations in the cytoplasm (supplementary material Fig. S3B). Bycontrast, cells that retained their flagellum showed a normal stainingpattern. The endoplasmic reticulum was analysed with an antibodyagainst the chaperone protein BiP, which normally decorates theperiphery of the nucleus and most of the cytoplasm (Bangs et al.,1993): a staining pattern reproduced in non-induced populations(supplementary material Fig. S3C). Staining was apparently normalin non-flagellated cells, but turned out to be stronger inmultinucleated cells (supplementary material Fig. S3D). To ensurethat these modifications were not due to general disorganisation ofmutant cells, the distribution of organelles unrelated to the endocyticnetwork such as the mitochondrion or the glycosomes was analysed.An antiserum recognising the glycolytic enzyme aldolase and themitochondrion tracer MitoTracker (not shown) were used for theanalysis. In both cases, normal staining patterns were observed inflagellated and non-flagellated cells (supplementary material Fig.S3E,F).

Non-flagellated cells show reduced ability for endocytosisThese multiple phenotypes raise the question whether the flagellarpocket is competent for endocytosis in IFTRNAi mutants. Receptor-mediated endocytosis cannot be traced easily in procyclictrypanosomes but fluid-phase endocytosis can be monitored byincubating cells with the lipophilic dye FM4-64 (Hall et al., 2005).Induced or non-induced cells were incubated at 4°C with FM4-64and subsequently heated at 27°C, which should result ininternalisation through the flagellar pocket and accumulation at theposterior region. This was observed in non-induced trypanosomes(quantification at Fig. 8C). In induced IFT172RNAi and IFT88RNAi

cells, those cells that still possessed a flagellum showed a similar

Fig. 8. The function of the flagellarpocket is altered in the absence of anormal flagellum in IFTRNAi cells.(A-D) Capture and accumulation ofthe lipophilic tracer FM4-64 (red) inthe indicated cell lines induced for48 hours. Cells were fixed andstained with DAPI (blue)immediately after washes. Whitearrows indicate non-flagellated cells.(C) Quantification of the amount ofFM4-64 signal in the indicated cells.For IFT172RNAi induced samples,counts for flagellated and non-flagellated cells from the same slideare presented. FP+++, bright signalat the posterior end of the cell;FP+/–, weak signal at the posteriorend; diffuse+++, bright signalthrough a large zone of the cell(always the posterior end forflagellated cells, more variable fornon-flagellated ones); diffuse+/–,weak signal through a large zone ofthe cell. n=50-120.

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bright signal, demonstrating that the flagellar pocket of the oldflagellum remains functional (Fig. 8A-D). Strikingly, the vastmajority of non-flagellated cells presented much less (but stilldetectible) fluorescence (Fig. 8A-C), which was often moredispersed and not restricted to the flagellar pocket area. Dividingcells were analysed at early stages of RNAi, when they possess anold flagellum but do not grow a new flagellum (Fig. 8D). FM4-64accumulation occurred normally and the tracer concentrated aroundtwo separate zones, each found in the vicinity of a kinetoplast. Thisshows that despite the absence of a normal flagellum, a new flagellarpocket can be formed, which is competent for endocytosis, at leastbefore the cell undergoes cytokinesis. Nonflagellated cells exhibita much-reduced capacity to capture tracers, showing that thepresence of the flagellum is required for maintaining a fullyfunctional flagellar pocket.

DiscussionThe flagellum contributes to structural development andorientation of the flagellar pocketOur data reveal that in the absence of elongation of the newflagellum upon silencing of distinct components of the IFTmachinery, a flagellar pocket develops close to the basal body andkinetoplast complex. However, this flagellar pocket exhibits adifferent structure compared with that in control cells or with theflagellar pocket associated with the old flagellum. First, transmissionelectron microscopy showed the presence of a shorter flagellarpocket in cells without a flagellum or of modified shape in caseswhere the flagellum was abnormally short. Second, the orientationof the FPC relative to the long axis of the cell was much morevariable compared with the well-defined positioning observed incontrol cells. Moreover, the structure of the collar consistentlylooked thinner or more irregular, as revealed upon detergentextraction. Third, the BILBO1 protein, the only molecular markerof the FPC, showed partially modified staining in cells with a shortor no flagellum.

Flagellum replication and flagellar pocket formation areintertwined processes that can be summarised as follows (Fig. 9).After basal body duplication, the new flagellum is constructed inthe large anterior side of the existing flagellar pocket (Absalon etal., 2008; Briggs et al., 2004; Grasse, 1961). The new flagellumcontains a significant amount of IFT-like amorphous material(Absalon et al., 2008; Grasse, 1961), a situation reminiscent of theshort new flagella of Chlamydomonas (Dentler, 2005). Flagellarmicrotubules elongate and the new flagellum grows in the sameflagellar pocket as the old flagellum. However, when the newflagellum exits from the cell body, it possesses its own flagellarpocket. Analysis of control trypanosomes failed to identify cells withtwo flagella emerging from the same flagellar pocket, suggestingthat the new flagellum acquires its own FPC before or as it emergesfrom the cell body. Transmission and scanning electron microscopyof detergent-extracted cells suggest that the collar of the new flagellarpocket is thinner compared with that of the old flagellum. At thisstage, the new flagellum is in a posterior position relative to the oldone, indicating that rotation of its basal body must have taken placeprior to formation of the flagellar pocket associated with the newflagellum. Elongation continues, the basal bodies migrate apartextensively, and the new flagellar pocket acquires associatedorganelles, such as the Golgi complex (He et al., 2004) or thelysosomes (Alexander et al., 2002). Endocytic/exocytic vesiclesbecome visible (Jeffries et al., 2001; Morgan et al., 2001) and FM4-64 uptake reveals endocytosis activity (Hall et al., 2005).

In cells that cannot assemble a new flagellum (no anterogradeIFT), duplication of the basal body complex takes place normally,at the expected anterior position. It is followed by formation of anew but shorter flagellar pocket with a less developed or structuredcollar. Basal body rotation and initial migration to a posteriorposition occur, showing that the flagellum is not required for theseprocesses. However, complete migration of the new basal bodytowards the posterior end is rarely observed (Absalon et al., 2007).In mutants of retrograde IFT, a short, dilated, flagellum is formedassociated with a flagellar pocket of an apparently normal size butof drastically modified shape. This implies at least two contributionsof the flagellum to flagellar pocket formation: (1) elongation,possibly by providing a supporting anchor and (2) shape definition,possibly by bringing specific proteins to the contact area betweenthe flagellar pocket and the flagellum membrane. Movement offlagellar membrane proteins by IFT has been shown in otherorganisms (Huang et al., 2007; Qin et al., 2005) and could beenvisaged for proteins linking the FPC to the flagellum membrane.Thus, flagellum elongation could be required for formation of afully functional FPC, as suggested by common phenotypes observedin IFTRNAi and BILBO1RNAi mutants (Bonhivers et al., 2008b), suchas accumulation of vesicles containing CRAM and disturbed p67labelling.

Another phenotype is the randomisation of flagellar pocketorientation in both types of IFT mutants. In normal cells, detergenttreatment reveals the FPC as a horseshoe-like structureperpendicular to the axis of the flagellum, with its posterior partslightly lifted above its point of emergence from the cell body (Fig.9). This configuration is visible by scanning electron microscopeobservation of whole cells, confirming that this defined positioningis not an artefact of detergent extraction. Given the role of forwardmotility in shifting surface-bound ligands to the flagellar pocket(Engstler et al., 2007), such a configuration could restrict movementbeyond the flagellar pocket and favour penetration in the flagellarpocket. This precise orientation is lost in both types of IFT mutantcells and the flagellar pocket is found in any position. Thedisorientation of the new flagellar pocket is already detected in cellsthat still possess the old flagellum, and could be due to the failedbasal body migration typical of IFT mutants (Absalon et al., 2007).

Perturbation of flagellar pocket functions in IFT mutantsStructural modifications of the flagellar pocket were accompaniedby several defects: reduced fluid-phase endocytosis, differentvesicular profile in the cytoplasm, presence of vesicles in the shortflagella and mislocalisation of CRAM that leaked out of the flagellarpocket and on to the surface of the short flagella of IFT140RNAi

cells. In wild-type cells, CRAM is abundant at the flagellar pocketclose to the base of the flagellum (Lee et al., 1990). Dissectionstudies revealed that the short cytoplasmic tail of CRAM is essentialfor retention in the flagellar pocket. In its absence, the truncatedprotein is allowed to diffuse along the length of the flagellum,indicating the existence of a molecular filter at the junction betweenthe flagellum and the flagellar pocket (Yang et al., 2000). Thismolecular filter is clearly less or not active in induced IFT140RNAi

cells, because CRAM is allowed to diffuse in the flagellarcompartment and on the surface of the flagellar sleeve. This suggeststhat proteins localised to the flagellum or flagellar pocket filter areaare missing or non-functional. An alternative explanation could bea direct involvement of retrograde IFT in recycling CRAM to thebase of the flagellum in normal cells, a feature that is not functionalin retrograde mutants. By contrast, the presence of CRAM at the

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rest of the cell surface is rarely observed, indicating that the physicaldistinction between the pellicular membrane and the flagellum andflagellar pocket membranes (Bastin et al., 2000) is still operating.

Endocytosis and exocytosis exclusively take place at the flagellarpocket. Fluid-phase endocytosis still takes place in non-flagellatedcells, in agreement with the presence of a flagellar pocket. However,it is much less efficient, which could be attributed to a combinationof several factors: (1) a smaller flagellar pocket surface; (2) a lowerdensity of proteins involved in endocytosis; (3) the absence offorward motility; and (4) the randomised orientation of the flagellarpocket. Reduced endocytosis probably results in supplementaryproblems in the balance with exocytosis, for example if the modifiedflagellar pocket is not able to cope with the arrival of exocyticvesicles. This could explain the formation of the flagellar sleeve(Davidge et al., 2006) that regularly looks like a succession ofvesicles in all nine IFTRNAi mutants examined to date (Absalon etal., 2008) (our unpublished data). The CRAM signal was much moreapparent in the cytoplasm, where it could accumulate at the levelof the ER (Yang et al., 2000). This could be due to the imbalanceof vesicle trafficking, if the same amount of CRAM is producedbut unable to properly insert in the flagellar pocket, thereforeaccumulating in the exocytic pathway.

The presence of vesicles in the flagellar pocket could be relatedto the emergence of vesicles with a double membrane found in the


Golgi and flagellar pocket regions, as suggested by their similarfrequency (~30%) (Figs 2 and 7). The origin of these vesicles isnot clear at this stage, but they seem to arise from the Golgi areawhere deformed vesicles have also been identified. Bending of suchvesicles would lead to formation of double-membrane vesicles,which in turn could fuse with the flagellar pocket membrane andhence deliver a single membrane vesicle in the flagellar pocketlumen. Excessive vesicles could be targeted for autophagy and bentvesicles might represent the initial stage of this process. Theproportion of sections showing concentric vesicles indicatingautophagy rose to 5-10% in IFTRNAi mutants compared with 1% inwild-type controls. However, a similar increase was noted for theaxonemal mutants PF16RNAi and PF20RNAi, which display flagellarpockets of normal size and shape (Branche et al., 2006), suggestingthat this phenomenon is not specific to defects in flagellar pocketfunction.

Alternatively, flagellar pocket vesicles could arise from directvesicle budding from the flagellar pocket membrane, possiblyrelated to the plasma membrane ‘blebs’ observed upon silencing ofWCB, a protein containing calcium-binding C2 domains that isassociated to the subpellicular microtubules of the corset (Bainesand Gull, 2007). Intriguingly, some flagellar proteins contain C2domains (Avidor-Reiss et al., 2004), although none of the IFTproteins studied here present such a motif.

Fig. 9. Diagram summarising the role of theflagellum in flagellar pocket formation. (A) Generalview of the monoflagellated trypanosome cell wherethe flagellar pocket area has been made transparent tovisualise flagellar pocket associated components. Forsimplicity, only the main organelles studied in thiswork have been represented. (B-D) Situation in wild-type cells with one flagellum (B), one old and onenew flagellum prior to microtubule assembly (C) andafter flagellar pocket duplication (D). (E) Situation ina nonflagellated cell where anterograde IFT wasinhibited. (F) Situation in a cell where a shortflagellum has been produced upon blockage ofretrograde transport. The internal region of theemerging flagellum is also shown. See text fordetails.Jo

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In conclusion, elongation of the new flagellum is not requiredfor flagellar pocket formation but is central to flagellar pocketstructural development and orientation. IFTRNAi mutants illustratetwo levels of this contribution: flagellar pocket elongation andflagellar pocket shape definition, with important functions in themolecular filter that separates the membrane of the flagellum fromthat of the flagellar pocket. Identification of the molecularcomponents of the flagellar pocket, such as the recent discovery ofBILBO1, should contribute to better understanding of thisremarkable and critical structure of trypanosomes. These resultscould be significant for other organisms, because the base of primarycilia in numerous mammalian cell types is positioned in aninvagination of the cell surface that is remarkably similar to theflagellar pocket (Han et al., 2008; Jensen et al., 2004; Sorokin, 1968).

Materials and MethodsTrypanosome cell lines and culturesAll cell lines used for this work were derivatives of strain 427 of T. brucei and culturedin SDM79 medium supplemented with hemin and 10% fetal calf serum. Cell linesDHC1bRNAi, IFT88RNAi (Kohl et al., 2003), IFT20RNAi (Absalon et al., 2007),IFT140RNAi and IFT172RNAi (Absalon et al., 2008) express double-stranded RNA fromtwo tetracycline-inducible T7 promoters facing each other in the pZJM vector (Wanget al., 2000), transformed in 29-13 cells that express the T7 RNA polymerase andthe tetracycline repressor (Wirtz et al., 1999). RNAi was induced by addition of 1μg tetracycline per ml of medium and fresh tetracycline was added at each cell dilution.

ImmunofluorescenceCells were washed, settled on poly-L-lysine-coated slides and fixed in 4%paraformaldehyde for 10 minutes (for anti-aldolase or anti-CRAM). Fixed cells werepermeabilised with 1% Nonidet P-40 for 10 minutes and samples were rinsed toremove excess detergent. Blocking was performed by incubation for 45-60 minutesin PBS containing 1% bovine serum albumin. Alternatively, cells or detergent-extracted cytoskeletons were washed, settled on poly-L-lysine-coated slides, fixed inmethanol at –20°C for 5 minutes and rehydrated in PBS (Mab22, anti-TBBC, anti-BiP, anti-p67). In all cases, slides were incubated with primary antibodies for 45-60minutes. Monoclonal mAb22 (dilution 1:5) is an IgM that detects an as yetunidentified antigen found at the exclusion zone of the tripartite attachment complexthat links the proximal zone of both the mature and the pro-basal body to the kinetoplast(Bonhivers et al., 2008a; Pradel et al., 2006). TBBC presence and localisation wasanalysed with a rabbit polyclonal antiserum (dilution 1:100) (Dilbeck et al., 1999).The anti-BILBO1 antibody was used as a marker of the FPC (IgM, dilution 1:2 inPBS after blocking in 0.5% BSA for 10 minutes, and revealed by a FITC-conjugatedsecondary antibody 1:100 in PBS, Sigma). Antibodies against p67 (1:200) and BiP(1:500) were used as markers for lysosomes (Alexander et al., 2002) and theendoplasmic reticulum (Bangs et al., 1993), respectively. An antiserum raised againstaldolase (dilution 1:4000) was used as marker of glycosomes (kind gift from PaulMichels, Christian de Duve Institute, Brussels, Belgium). Slides were washed in PBSand incubated with the appropriate secondary antibodies coupled to Alexa Fluor 488(Invitrogen), Cy3 or Cy5 (Jackson). Slides were stained with DAPI for visualisationof kinetoplast and nuclear DNA content.

For detergent treatment, cells were settled on poly-L-lysine-coated slides andexposed to various concentrations of Nonidet P-40 in spindle stabilisation buffer madeof 4 M glycerol, 10 mM PIPES (pH 6.5), 10 mM MgCl2 and 5 mM EGTA (Roobolet al., 1984).

For FM4-64 uptake, induced and non-induced RNAi cell lines were grown at adensity of 1-5�106 cells per ml. A total of 107 cells were washed and incubated with40 μM FM4-64 for 20 minutes at 4°C or at 27°C and treated as described (Hall etal., 2005).

Slides were observed with a DMR Leica microscope and images were capturedwith a Cool Snap HQ camera (Roper Scientific). Alternatively, slides were also viewedon a DMI4000 Leica microscope and images were acquired with a Retiga-SRV camera(Q-Imaging). Images were analysed using the IPLab Spectrum 3.9 software(Scanalytics & BD Biosciences) or Image J (NIH).

Electron microscopyCell fixation, embedding and sectioning for transmission electron microscopy of wholecells from wild-type and induced PF20RNAi, DHC1bRNAi, IFT88RNAi and IFT172RNAi

samples was carried out as described previously (Branche et al., 2006). Cells fromthe IFT140RNAi cell line were treated similarly except that tannic acid was not includedand that dehydrated samples were embedded in Epon resin followed by polymerisationfor 48 hours at 60°C. For scanning electron microscopy of whole cells, samples wereprepared and analysed as described previously (Absalon et al., 2007). For immunogolddetection with the anti-CRAM antibody, 107 cells were washed twice before fixationfor 45 minutes in 4% paraformaldehyde and 0.1% glutaraldehyde, followed by two

5 minute washes and neutralisation in PBS containing 50 mM NH4Cl for 30 minutes.Coverslips were incubated in PBS containing 1% BSA for blocking and incubatedwith the anti-CRAM (dilution 1:150) in PBS with 0.1% BSA for 45 minutes. Afterthree washes in the same buffer, bound antibodies were labelled by addition of ProteinAG conjugated to gold particles of 20 nm diameter (1:70) in PBS with 1% BSA.Coverslips were then prepared for scanning electron microscopy analysis accordingto standard procedures (Absalon et al., 2007), except that metallisation was carriedout with carbon instead of gold-palladium.

Cells were treated with 0.1-1% Triton X-100 at 4°C in PBS for 10 minutes to stripthe plasma membrane and visualise the FPC. Samples were washed twice in PBS,fixed in glutaraldehyde and processed for scanning electron microscopy in standardconditions (Absalon et al., 2007).

We thank Gwéndola Doré for assistance in plasmid DNA preparationand in preliminary immunofluorescence experiments; Mark Field andBelinda Hall for setting up the FM4-64 assay and for variousdiscussions, Derek Nolan for discussions about the flagellar pocketphenotype, Markus Engstler for critical reading of the manuscript,Etienne Pays, Jay Bangs and Paul Michels for providing severalantibodies. We acknowledge the electron microscopy departments ofthe Muséum National d’Histoire Naturelle and of the Pasteur Institutefor providing access to their equipment, as well as advice from Marie-Christine Prévost and Stéphanie Guadagnini. This work was fundedby the CNRS, by a Programme Protéomique et Génie des Protéines(D.R. and P.B.), an ACI grant in Development Biology, by a GIS granton rare genetic diseases (P.B.) and by Sanofi-Aventis. S.A. was fundedby a MRT fellowship, by an FRM doctoral fellowship and by a Pasteur-Weizman fellowship. J.B. is funded by a MRT fellowship.

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