The specificity of the anti-GPI-PLC antibody was validated by Western blotting following SDS-PAGE. Coomassie blue staining revealed little if any detectable difference between wild-type and GPI-PLC null mutant cells (Figure 1, panel A, image 1, tracks +/+ and −/− respectively). However, the antibody detected a single protein band at approximately 39 kDa in wild-type cells, which was absent in the GPI-PLC null mutant cells (Figure 1, panel A, image 2, tracks +/+ and −/− respectively).

The GPI-PLC in wild type bloodstream forms of T. brucei was immunoprecipitated using the anti-GPI-PLC antibody after extraction of cells with a mild neutral detergent mixture (see Methods). The immunoprecipitates were subjected to SDS-PAGE followed by Western blotting. The Coomassie stained gel revealed the presence of several bands. The two most prominent bands corresponded to the heavy chain (M_r 55000) and the light chain (M_r 23000) of IgG and a sharp band at 39000 corresponded to the M_r of the GPI-PLC (Figure 1, panel B, image 1, track b) which was absent from the immunosupernatant (Figure 1, panel B, image 1, track a). Several other weaker bands at higher M_r than the heavy chain of IgG were also present in the immunoprecipitate. When the bands from the immunoprecipitate in a replica gel were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with the anti-GPI-PLC antibody, only the band corresponding to the GPI-PLC was detected (Figure 1, panel B, image 2, track b). This band was absent from the immunosupernatant (Figure 1, panel B, image 2, track a). The Coomassie blue stained band in a gel from a parallel experiment, corresponding to the 39 kDa protein band and recognized by the anti-GPI-PLC antibody in the Western blot, was cut from the gel, eluted, digested with trypsin and subjected to MALDI-TOF MS. The peptides detected were all derived from the GPI-PLC (Figure 1, panel B, image 3) with 31% sequence coverage, distributed uniformly throughout the protein.

In addition, MALDI-TOF MS also correctly identified the peptides in both portions of the intact GPI-PLC-MBP fusion protein and in the separated GPI-PLC and MBP after cleavage of the fusion protein (Text S1 and Figure S1).

Immunoprecipitates were prepared from whole wild-type surface radio-iodinated Molteno Institute antigenic type (MITat) 1.2 cells and analysed by SDS-PAGE and autoradiography. No radio-labelled GPI-PLC was immunoprecipitated when preimmune IgGs were used (Figure 2, track 1). In this case only a very small amount of contaminating VSG was observed in the immunoprecipitate, which probably results from the large excess (approx. 200 fold) of VSG over that of the GPI-PLC in wild-type cells. No other proteins were detected when preimmune IgGs were used. In a separate but similar experiment the autoradiogram of the SDS-PAGE gel, containing the proteins in the anti-GPI-PLC immunoprecipitates of wild-type cells, displayed a strong band corresponding to the GPI-PLC as well as strong bands corresponding to several other proteins (Figure 2, track 3). However, when the same experiment was performed using GPI-PLC null-mutant cells, there was no band corresponding to the GPI-PLC in the autoradiogram (Figure 2, track 2). In addition, surface labelling of cells did not label the internal protein, tubulin (Text S1 and Figure S2). These experiments strongly suggest that the GPI-PLC is exposed to the external face of the plasma membrane.

The GPI-PLC was detected by immunofluorescence using the anti-GPI-PLC antibody in wild-type bloodstream forms (Figure 3, panel A) and was absent in GPI-PLC^−/− bloodstream forms (Figure 3, panel B) and in procyclic forms (Figure 3, panel C). The universal presence of the GPI-PLC was particularly striking in all wild type bloodstream form cells when viewed at low power magnification where many cells are found in the single field of view (Figure 3, panel D). Likewise the absence of the GPI-PLC in GPI-PLC null mutant bloodstream forms was obvious in all cells within the population in low magnification views (Figure 3, panel E). The distribution of the GPI-PLC in wild type cells was universally found in a single, patchy, linear array, along the line of flagellar attachment (Figure 3, panel A). It was never found in cytoplasmic vesicles, in the regions usually occupied by either the endoplasmic reticulum or the glycosomes. In addition it was never observed uniformly or even in a patchy distribution throughout the whole of the plasma membrane itself.

In natural infections, parasitaemias are generally pleomorphic with rapidly dividing slender forms that predominate in the ascending parasitaemia and short-stumpy forms in the descending parasitaemia [26],[27],[28]. The GPI-PLC was found to have the same pattern of fluorescence in both long-slender forms in the monomorphic strain, MITat 1.2 and short-stumpy forms in the very pleomorphic strain, S-42, but with the patchy nature of the linear array particularly striking in these short-stumpy forms (compare Figure 3, panels G and H with Figure 3, panel A).

In order to eliminate any possibility that the location of the GPI-PLC, determined by fluorescent antibody staining of cells, was due to an artefact, perhaps caused by reaction with a non-GPI-PLC epitope in bloodstream forms of T. brucei under the conditions employed for immunofluorescence, a gene encoding the GPI-PLC itself, tagged with yellow fluorecent protein (YFP), was introduced into cells and stably expressed. Its presence was detected by direct fluorescence of the YFP portion of the fusion protein itself. The location of the GPI-PLC was found by this alternate method to be identical to that determined by anti-GPI-PLC antibody staining. The GPI-PLC-YFP fusion protein was located mainly in the usual patchy linear array along the flagellum adjacent to the flagellar attachment zone (Figure 3I).

A much smaller fraction of the fluorescence was found within one or two small endo-membrane-bound cellular compartments (Figure 3I marked with arrowheads), anterior to the flagellar pocket but very close to the pellicular microtubules and the plasma membrane. The identity of this second compartment remains to be determined.

This second compartment has an interresting morphology. It is roughly flask-shaped with either a round or stocking-like body and a single neck that at one end is directed towards the plasma membrane. In some views the neck appears to be physically joined at its end to the plasma membrane. In a few cells this same location and morphology were also found by immunofluorescence (Figure 4A-2, see arrowheads). It may be significant that this structure and location were not found in all cells, whereas the linear array of the GPI-PLC on the flagellar membrane was a constant finding in all cells. For example, this second compartment may be present in only some stage(s) of the cell cycle. Furthermore, this observation together with its location and attachment to the plasma membrane may suggest that it is a structure involved in the assembly of the proteins destined to form the new flagellar attachment zone. In support of this possibility was the additional observation, using fluorescence-tagged antibodies, that this second structure also contained the FAZ1 protein, even though it was not entirely co-localized with the GPI-PLC (small arrows within the small lower insets in Figure 4 panels C-2, C-3 & C-4).

The VSG is the predominant surface protein in bloodstream form trypanosomes and forms the outermost surface of these cells, even shielding other exterior proteins. The VSG was evenly distributed over the entire surface of the trypanosome as reported previously by others [10],[29] and was visible when fixed, at either 0°C or 37°C in both the presence and absence of triton X-100 (Figure 5, panels A–D).

Tubulin is the major protein of microtubules in the trypanosomal cytoskeleton and is found as a pellicular array attached to the cytoplasmic face of the plasma membrane and as the flagellar axoneme enclosed by the flagellar plasma membrane. Therefore, mouse monoclonal IgG anti-α-tubulin (DM1A) antibody [30], followed by Alexa 488-conjugated anti-mouse IgG, identifies both the position of the inner-most surface of the plasma membrane of the cell body, as well as the axonemal portion of the flagellar compartment. However, this antibody combination detected tubulin in trypanosomes only when 0.1% triton X-100 was present during the blocking and antibody treatment steps regardless of whether trypanosomes were previously fixed at either 0°C or 37°C (Figure 5, panels A & C) and failed to detect tubulin in the absence of triton X-100 (Figure 5, panels B & D). The distribution of tubulin was found to produce a uniform fluorescence from FITC-labelled anti-tubulin along the inner surface of the cell body as well as along the central portion of the flagellum.

Invariant Surface Glycoprotein70 (ISG-70) is an externally disposed integral membrane glycoprotein, present only in bloodstream forms, and is found in a patchy distribution over the entire surface of the plasma membrane covering both the cell body and flagellum. The VSG from a number of different variants masks the presence of ISG-70 in live but not in fixed cells [31]. Probing fixed trypanosomes with purified polyclonal rabbit anti-ISG-70 primary antibody followed by Cy-3-labelled goat anti-rabbit IgG secondary antibody identifies the area of the outer leaflet of the plasma membrane shielded by the VSG. ISG-70 was found distributed over the entire surface in an irregular, patchy pattern when cells were fixed at 0°C, in both the presence and absence of triton X-100 (Figure 5, panels A & B). However, fluorescence was absent when trypanosomes were fixed at 37°C (Figure 5, panels C & D) in both the presence and absence of triton X-100.

The GPI-PLC was detected by rabbit polyclonal IgG anti-GPI-PLC, followed by Cy-3-conjugated goat anti-rabbit IgG, in cells fixed at 0°C (Figure 5, panels A and B), while fixation at 37°C resulted in no detectable fluorescence (Figure 5, panels C and D). Detection of the GPI-PLC was insensitive to the presence or absence of detergent. Clearly, the ability to detect the GPI-PLC follows the pattern displayed by ISG-70 with respect to temperature and detergent and does not resemble the pattern shown by either the VSG or tubulin, suggesting that the GPI-PLC is located in a restricted portion of the outer leaflet of the plasma membrane and at least partially shielded by the VSG in the same manner as ISG-70

In order to verify that the GPI-PLC was not localized to the inner face of the plasma membrane, its location with respect to tubulin was investigated. The distribution of tubulin was found to produce an intense fluorescence due to the large numbers and regular disposition of the subpellicular microtubules. The flagellum was seen as a fluorescent, wavy line along the side of the cell body, due to the microtubules of the flagellar axoneme (Figure 4, panel A, images 3 & 4). The GPI-PLC was found distributed between these two regions of microtubules lying closer to the flagellar axoneme than to the cell body (Figure 4, panel A, images 2 & 4) and at no point were the two fluorescent dyes co-localized. Furthermore, the GPI-PLC was distributed along the line of the flagellum, indicating that the GPI-PLC was not localized to the cell body but, rather, to the flagellar compartment. A small fraction of the GPI-PLC was also seen in a few cells within an endo-membrane-bound space close to the inner surface of the cell anterior to the flagellar pocket (see arrowheads in Figure 4, panel A, image 2).

The paraflagellar rod is a large structure found within the flagellum of bloodstream form trypanosomes, extending from the point where the flagellum exits the flagellar pocket, running alongside the axoneme of the flagellum to its distal tip, and is physically connected to the axoneme via fibres attaching the proximal domain to microtubule doublets 4 through 7 [32]. The crescent shape of the paraflagellar rod and its location between the axoneme and the flagellar attachment zone in trypanosomes prompted us to determine the localization of the GPI-PLC simultaneously with that of the paraflagellar rod.

The pattern of staining using anti-PFR-A confirmed past observations [32],[33]. The paraflagellar rod appeared as a slightly curved thick linear rod within the flagellar compartment adjacent to the axoneme, stretching from the lip of the flagellar pocket to the distal tip of the free flagellum (Figure 4, panel B, images 3 & 4). The GPI-PLC was detected in the same cells by treatment with rabbit IgG anti-GPI-PLC, followed by Cy-3-conjugated goat anti-rabbit IgG, and was found in a patchy, linear array, beginning at a point close to the lip of the flagellar pocket but slightly more distal than the proximal end of the paraflagellar rod, to an area just posterior to the tip of the free flagellum far beyond the end of the cell body but ending before the distal tip of the paraflagellar rod (Figure 4, panel B, images 2 & 4 and image 4 inset). The GPI-PLC was located adjacent to the paraflagellar rod on the side of the rod facing the cell body in the region where the flagellum is attached to the cell body (Figure 4, panel B, images 2 & 4). However, the localization of the GPI-PLC was clearly distinct from that of the paraflagellar rod. In fact the merged image (Figure 4, panel B image 4) shows that the two proteins can be resolved into two non-overlapping linear structures. This distribution is particularly striking when the flagellum appears to cross from one side of the cell body to the other side of the cell body; e.g. when the flagellum, running along the top of the cell body falls towards one or the other side of the cell body. In each case the paraflagellar rod lies furthest from the cell body and the GPI-PLC lies closest to the cell body (Figure 4, panel B, image 4). This result indicates that the GPI-PLC is not located in the same position as the paraflagellar rod but, rather, lies in the region between the paraflagellar rod and the cell body. Furthermore, the GPI-PLC (Figure 4, upper arrows in both panel B image 4 and in inset) does not extend along the free flagellum as far as the paraflagellar rod (Figure 4, lower arrows in both panel B, image 4 and in inset).

The flagellar attachment zone (FAZ) is found in the cytoplasmic compartment of the cell body, lying adjacent to the paraflagellar rod, which is in the flagellar compartment, in bloodstream form trypanosomes [33] and is composed of a number of proteins. These published images prompted us to determine whether the GPI-PLC was located in the same compartment as the FAZ, because we found the GPI-PLC distributed immediately adjacent to the PFR on the cell body side of PFR (Figure 4, panel B, image 4), which is certainly close to the FAZ region. The fluorescence observed with mouse monoclonal IgG anti-FAZ1 protein antibodies (L3B2) followed by Alexa 488-conjugated goat anti-mouse IgG antibody (Figure 4 panel C images 3 and 4) was found in a region between the cell body and the flagellum (Figure 4, panel C, image 4 compare with Figure 4, panel C, image 5). The GPI-PLC was distributed in the usual patchy linear array along the outer side of the FAZ (Figure 4, panel C, image 4). The distribution of both the FAZ and the GPI-PLC begin close to the flagellar pocket, just anterior to the kinetoplast. However, the GPI-PLC (Figure 4, panel C, image 4, upper arrow and inset, upper arrow) extends beyond the distal tip of the FAZ (Figure 4, panel C, image 4, lower arrow and inset, lower arrow) into the region of the free flagellum. Even in the regions where both proteins are found, it is apparent that the GPI-PLC does not co-localize with the FAZ1 protein (Figure 4, panel C, image 4, arrowhead). The GPI-PLC was located immediately adjacent to the outer side of the FAZ on the same side as the flagellum. Combining the information from the results in Figure 4, panels B and C) one can conclude that the GPI-PLC lies between the flagellar attachment zone (FAZ) and the paraflagellar rod (PFR). Therefore, when the flagellum lies on its side, the FAZ is closest to the cell body and the GPI-PLC is furthest from the cell body (Figure 4, panel C, image 4). The only structures that lie between the FAZ and the paraflagellar rod are the plasma membrane of the cell body and the flagellar plasma membrane. However, the GPI-PLC only co-localizes with the proteins of the flagellar plasma membrane and not with the proteins of the plasma membrane surrounding the cell body (Figure 4, panel D, image 4). Consequently, we conclude that the GPI-PLC is located in the flagellar plasma membrane.

Figure 6, panels A & B are presented in order to provide additional evidence that firstly, the GPI-PLC and the FAZ1 protein are not in the same location and secondly, that the GPI-PLC is localized to the flagellar membrane of bloodstream form trypanosomes, while the FAZ1 protein remains with the cell body. The trypanosomes shown in Figure 6 have been selected because they are slightly damaged with their flagellum and surrounding membrane torn from their attachment to the plasma membrane of the cell body. These images distinguish clearly between the distribution of the GPI-PLC and the FAZ1 protein. The FAZ1 protein remains attached to the cell body (Figure 6, panel A, images 2, 4 and 5) while the GPI-PLC remains aligned along the flagellum (Figure 6, panel B, images 2, 4 and 5). Furthermore, the GPI-PLC was not distributed over all of the flagellar membrane. Rather, it occupied a position on the flagellar membrane immediately adjacent to the cell body (Figure 6, panel B, images 2, 4 and 5).

The plasma membrane of bloodstream forms of T. brucei consists of three distinct but contiguous domains: the pellicular membrane, the flagellar membrane and the flagellar pocket membrane. Labeling trypanosomes with a Cy-3-dye conjugated to a maleimide reactive group, acts as a membrane protein marker and detects all three plasma membrane domains without labeling any internal membranes. It appears that the two negatively charged sulphonate groups and the positively charged quaternary nitrogen on the dye are sufficient to prevent entry of the dye into the cell, particularly in the presence of a negative plasma membrane potential, which excludes net negatively charged molecules lacking a specific energy-coupled transport system, e.g. the thiocyanate anion [34]. The cellular distribution of the GPI-PLC is shown in Figure 4, panel D, images 2, 4 and 5 in an actively dividing and, consequently, biflagellate cell. It is aligned along the length of both flagella. The Cy-3 dye has labeled both the plasma membrane of the cell body and the flagellar membrane (Figure 4, panel D, images 3, 4 and 5). When the images of the GPI-PLC and the plasma membrane fluorescence are merged the resulting image suggests that the GPI-PLC is located on the membrane of both flagella (Figure 4, panel D, image 4). The patchy linear distribution of the GPI-PLC localizes along the exterior portion of the membrane of the flagellum and is nowhere near the membrane of the cell body. Consequently, it appeared likely that the GPI-PLC was associated with the flagellar membrane. A similar result was obtained using the membrane surface lipid probe, fluorescein-derivatized dihexadecanoyl-glycerophosphoethanol amine [35] in conjunction with rabbit polyclonal IgG anti-GPI-PLC antibody and a Cy-3 conjugated goat anti-rabbit IgG secondary antibody (data not shown).

An experiment was conducted to determine whether the activated GPI-PLC moved away from its previous position in the unactivated state. We have found that incubation of bloodstream form trypanosomes in the absence of glucose but in the presence of 2-deoxyglucose (10 mM) to de-energize cells and, further, in the presence of hyperosmotic phosphate - sucrose buffer to prevent cell rupture, activates the GPI-PLC and quantitatively releases the VSG over a 45 min time course. The distribution of the GPI-PLC in trypanosomes that have lost their VSG but are still intact was the same as it was in fully energized trypanosomes (Compare Figure 7, panel A, images 2 & 3 with panel B, images 2 & 3). These results suggest that the GPI-PLC does not move from its location in the flagellar membrane in order to cleave the GPI anchor of the VSG. In addition to the confocal microscopic analysis, protein release from the cell surface was measured (Figure 7, panel C) and the released protein analyzed by SDS-PAGE and western blotting (Figure 7, panel D). These results establish that the VSG was released from de-energized trypanosomes with a half time of 19 min (Figure 7, panel C), and converted from mVSG to sVSG as indicated by the paradoxical shift in apparent molecular weight [36] to an higher value (compare Figure 7, panel D, image 1, lanes a & b). Furthermore, conversion of the mVSG to the sVSG resulted in exposure of the CRD epitope on the sVSG, indicating that the release was due to cleavage of the VSG anchor by the GPI-PLC (compare Figure 7, panel D, image 2, lanes a & b). Consequently, release of the VSG occurred when the VSG diffused within the plane of the membrane to the position of the activated GPI-PLC and the activated GPI-PLC did not move to the position of each molecule of the VSG in turn.

All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committee.

Anti-GPI-PLC IgG and anti-ISG-70 were prepared by the popliteal lymph node route in rabbits [54] using either recombinant GPI-PLC [55] or purified ISG-70 [31]. After 3 subcutaneous boosts at 2-week intervals crude anti-sera was prepared, divided into 3 ml samples, frozen and stored at −20°C. Rabbit IgG anti-MITat 1.2 VSG was prepared by the procedure of O'Beirne & Voorheis [56]. All crude anti-sera were purified by elution from a protein A-Sepharose column [57]. Rabbit anti-CRD IgG was prepared by immunization with soluble ILTat 1.21 VSG. Cross-reacting antibodies were then eluted after absorption to MITat 1.6 VSG coupled to cyanogens bromide (CNBr)-activated Sepharose [58]. Anti-PFR-A and anti-FAZ1 antibody (L3B2) were kind gifts from Prof. Keith Gull (Sir William Dunn School of Pathology, University of Oxford, England). Anti-α-tubulin IgG (Clone DM1A) was obtained from Sigma. Alexa 488-labeled secondary antibodies were from Invitrogen (Molecular Probes). Cy-3-labeled secondary antibodies were from Jackson Immunoresearch.

For immunoprecipitation, protein-A purified rabbit anti-GPI-PLC IgG (156 µg in 0.02 ml) was bound to Protein A-Sepharose beads (0.02 ml; 50% v/v; in PBS buffer, pH 7.5, 136 mM NaCl, 3 mM KCl, 16 mM Na₂HPO₄, 3 mM KH₂PO₄) by incubation with rotational mixing overnight at 4°C. The protein A-Sepharose beads with attached antibody were washed twice with PBS buffer, pH 7.5, containing 15 mM sodium azide and once with lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% NP-40, 0.5% CHAPS, containing 0.1 mM tosyl-lysine chloromethyl-ketone and 0.3 mM phenylmethylsulphonylfluoride) and finally resuspended to 0.04 ml (∼50% v/v slurry of beads) in lysis buffer.

Both alleles of the GPI-PLC gene in the Molteno Institute trypanosomal antigenic repertoire (MITar) serodeme were deleted using two constructs in a manner similar to that used previously for the AnTar serodeme: first, with pLN [44] and second, with pLH, which was generated by replacing the GPI-PLC gene in p1172kxL with a hygromycin cassette as a KpnI-Xbal fragment [44]. Bloodstream forms of the MITar serodeme, derived originally from Lister 427 [59] were grown in culture at 37°C in HMI-9 medium [60], containing 10% (v/v) heat inactivated foetal bovine serum. These cells were electroporated twice in the presence of BglI-linearized DNA. First, the cells were brought to hygromycin resistance using 5 µg pLH in a standard electroporation [44]. Second, after selection using hygromycin (5 µg/ml), the uncloned population was electroporated in the presence of 5 µg pLN. A doubly resistant population was selected in the presence of both hygromycin and G418 (2.5 µg/ml) and then cloned by limiting dilution. The cloned population stably expressed MITat 1.2 VSG and was negative for expression of GPI-PLC by Western blotting.

The GPI-PLC was expressed as a fusion protein in frame with YFP at the C-terminus by modifying the endogenous locus in the GPI-PLC+/− heterozygote using the method described in [61]. After transfection, cell lines were selected for blasticidin resistance and the resulting fluorescent clones analysed. All of the several clones isolated gave similar patterns of fluorescence. For microscopy, ∼1×10⁶ log phase bloodstream form trypanosomes were recovered by centrifugation and resuspended in 10 µl of 0.02% formaldehyde in HMI-9 without serum at 25°C.

The gene encoding the GPI-PLC was amplified by PCR, using the ‘Expand’ high fidelity DNA polymerase system, from genomic DNA derived from T. brucei MITat 1.1 using a forward primer (5′-AGG GAT CCT TTG GTG TAA AGT GGT CAC CGC AG-3′) containing a BamH1 restriction site (underlined) and a reverse primer (5′-AGC AAC TCG AGT TAT GAC CTT GCG GTT TGG TTG GT-3′) containing an XHO1 restriction site (underlined) and then gel purified. This purified DNA was ligated into the lacZa gene within the cloning site of the similarly restricted, gel purified pMBP-parallel2 vector downstream of the malE gene, using T4 DNA ligase. The vector itself was the kind gift of Dr. Amir Khan, School of Biochemistry and Immunology, Trinity College Dublin and had been constructed by replacing the protease site in the linker fragment of a pMAL-c2 vector (New England Biolabs) with a tobacco etch virus (TEV) cleavage site. The sequence of the purified cloned product was found to be correct by DNA sequencing. The pMBP vector containing the GPI-PLC was transformed into E. coli BL21(DE3), expressed following addition of IPTG, and the fusion protein purified first by binding to an amylose resin column, eluted in the presence of maltose (10 mM), and then by chromatography on a Sephacryl S-200 column.

Growth, isolation and counting of bloodstream forms of T. brucei (MITat 1.1) were performed as described previously [62].

Bloodstream forms of T. brucei (10⁸ cells) were surface radioiodinated (1 mCi carrier-free sodium ¹²⁵Iodide) using glucose oxidase (0.66 units) and lactoperoxidase (0.165 units) for 10 min at 25°C as described previously [31] and surface biotinylated (5×10⁷ cells) using sulfo-N-hydroxy-succinamide long chain spacer arm (NHS-LC)-biotin (0.3 mg) in a final volume of 1.1 ml for 20 min at 0–4°C essentially as described by Gruszynski et al. [23].

Trypanosomes (5×10⁸ cells/ml) were washed twice in TSB buffer (44 mM NaCl, 5 mM KCl, 3 mM NaH₂PO₄, 118 mM sucrose, 10 mM glucose and 0.2 mM adenosine, pH 8). The cell pellet was resuspended in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% NP-40, 0.5% CHAPS, containing 0.1 mM tosyl-lysine chloromethyl-ketone and 0.3 mM phenylmethylsulphonylfluoride) and incubated for 30 min on ice. Cell lysates were centrifuged at 15,000 g for 15 min and the supernatant collected. GPI-PLC was immunoprecipitated with anti-GPI-PLC IgG bound to protein A-Sepharose beads. The immune complexes were removed from the beads by boiling for 2 min in SDS and their constituent proteins separated by SDS-PAGE and stained with Coomassie blue. The protein band corresponding to the GPI-PLC (∼39 kDa) was cut out, the protein passively eluted and concentrated by a minor modification of the method of Wessel and Flugge [63], digested with trypsin and analyzed by MALDI-TOF mass spectrometry. All MS data were acquired using a Voyager-DE PRO MALDI-TOF mass spectrometer equipped with a nitrogen laser.

Trypanosomes (5×10⁷/ml) were incubated (15 min) in PBS buffer (NaCl, 136 mM; KCl, 3 mM; Na₂HPO₄, 16 mM; sucrose, 40 mM; glucose, 10 mM; adenosine, 0.1 mM; pH 7.5) at 37°C in a final volume of 10 ml with constant gentle stirring to allow any adsorbed serum proteins to be removed by endocytosis. The incubation was terminated by adding an excess volume (5×) of ice-cold PBS buffer and centrifuging (9,000 g×10 seconds at 4°C) in a minifuge. The pellets were resuspended in ice-cold iso-osmotic phosphate buffer (Na₂HPO₄, 20 mM; sucrose, 360 mM; pH 7.5) containing protease inhibitors (0.3 mM phenylmethylsulphonylfluoride, 0.1 mM tosyl-lysine chloromethyl-ketone, 0.1% (w/v) leupeptin & 0.2% (w/v) pepstatin), at a final concentration of 5×10⁸ cells/ml. The cells (5×10⁸ cells) were then diluted into a second phosphate buffer without glucose (Na₂HPO₄, 20 mM; sucrose, 360 mM; pH 8.0) containing the same protease inhibitors and 2-deoxy-D-glucose (10 mM) at 37°C to give a final cell concentration of 5×10⁷/ml and incubated at 37°C for 45 min. The incubation was terminated by a 10-fold dilution into ice-cold phosphate buffer (Na₂HPO₄, 20 mM; sucrose, 360 mM; pH 7.5) and the cells in samples of the suspension collected by centrifugation. The supernatants containing the released VSG were concentrated by Vivaspin membrane ultra filters (5 kDa cut-off). Both the cell pellets and the supernatants were subjected to SDS-PAGE and subsequent western blotting. For confocal microscopy, cells were fixed for 10 min at 0°C by mixing with equal volumes of fixative in the same buffer solution at a final concentration of paraformaldehyde of 3% (w/v).

Bloodstream form trypanosomes (5×10⁷ viable cells/ml) were resuspended in PBS buffer and incubated at 37°C for 20 min with constant gentle stirring and then a first set of samples were removed and fixed for 10 min at 37°C with 3% (w/v) paraformaldehyde final concentration. A second set of samples were removed and cold quenched immediately by adding an excess (5×) of ice-cold PBS buffer, centrifuged (11,000 g, 2 min, 4°C) and the cells resuspended in PBS buffer and subsequently fixed for 10 min at 0°C with 3% (w/v) paraformaldehyde. Procyclic form trypanosomes were fixed for 30 min at 0°C. Following fixation, cells were washed twice in PBS buffer containing sodium azide (15 mM) before allowing the cells to settle on poly-L-lysine coated coverslips surrounded by thin nail varnish dams. Adherent cells were blocked with PBS containing 5% (w/v) BSA and 0.1 M methylamine, +/−0.1% (w/v) triton x-100 for 1 hr at room temperature. Cover slips with adherent blocked cells were incubated with the primary antibody in PBS containing 5% (w/v) BSA, +/−0.1% triton X-100 overnight at room temperature, followed by incubation with the secondary antibody in 5% BSA for 1 hr at room temperature. After two washes with PBS, the coverslips were mounted onto slides and sealed. The mounting media was 50% glycerol in PBS containing 4% (w/v) propylgallate anti-quenching agent. The DNA stain, Hoechst 33342, was added to the mounting media at 0.1 µg/ml to visualize the nucleus and kinetoplast.

For visualization of the paraflagellar rod and the FAZ, cells were settled on poly-L-lysine coverslips and fixed with cold methanol (−20°C) for 10 min, rehydrated with PBS and blocked with 5% (w/v) bovine serum albumin in PBS for 1 hr at room temperature as described by Sherwin et al. [64].

For labelling the protein in the outer leaflet of the plasma membrane of bloodstream forms of T. brucei, cells (5×10⁷ cells/ml) were incubated for 5 min at 30°C with a Cy-3 protein-reactive cyanine fluor (Amersham Cat. No. PA23001) that contains two negatively charged sulphonate groups and one positively charged quaternary nitrogen to prevent cell entry. Cells were then cold-quenched with 5 volumes of ice-cold PBS buffer and fixed at 0°C for 10 min with 3% (w/v) paraformaldehyde.

Primary antibodies and dilutions used were rabbit anti-GPI-PLC at 1∶1000, rabbit anti-VSG at 1∶1000, rabbit anti-ISG-70 at 1∶500, mouse anti-tubulin at 1∶350, mouse anti-PFR-A at 1∶50 and mouse anti-FAZ1 (L3B2) at 1∶5. Alexa 488 and Cy-3 -labeled secondaries were used at 1∶1000.

The GPI-PLC was probed with purified polyclonal rabbit anti-GPI-PLC IgG primary antibody. Tubulin was probed with mouse monoclonal anti-α-tubulin primary antibody (clone DMIA). The paraflagellar rod was probed with mouse monoclonal anti-paraflagellar rod-A IgG primary antibody [33]. The FAZ1 protein was probed with mouse monoclonal anti-FAZ1 IgG primary L3B2 antibody [33]. MITat 1.2 VSG was probed with purified rabbit polyclonal anti-MITat 1.2 VSG primary antibody. ISG-70 was probed with purified rabbit polyclonal anti-ISG-70 IgG antibody. All mouse monoclonal IgG primary antibodies were visualized using Alexa-488-conjugated goat anti-mouse IgG secondary antibody. All purified rabbit polyclonal IgG primary antibodies were visualized with Cy-3-conjugated goat anti-rabbit IgG secondary antibody.

Microscopy was performed on an Olympus FV1000 laser scanning Confocal Microscope, using an UPlanSAPO 60×/1.35 NA oil objective. Brightness and contrast adjustments were made to the whole digital image files and cropping images to size were both performed using Photoshop 9 (Adobe). Digital blind deconvolution of multichannel 3D microscopic images was performed with Bitplane Autodeblur software. Cells were digitally sectioned by confocal laser scanning fluorescence microscopy at 0.3 µm per slice and the composite Z-stack of all sections is shown in each figure.