Figure 1.
Specificity of the anti-GPI-PLC antibody for the GPI-PLC.
Proteins in both wild type GPI-PLC+/+ and GPI-PLC−/− null mutant trypanosomes were separated by SDS-PAGE on 15% (w/v) polyacrylamide gels and stained with Coomassie blue (panel A, image 1). The protein bands on replica gels were transferred to a PVDF membrane and probed with 3.9 µg of rabbit IgG anti-GPI-PLC primary antibody, followed by goat anti-rabbit IgG secondary antibody (panel A, image 2). The GPI-PLC was specifically immunoprecipitated from detergent lysates of whole bloodstream forms of T. brucei and was subjected to SDS-PAGE (panel B, image 1) and western blotting (panel B, image 2). In a parallel experiment the indicated 39 kDa protein band was cut from the gel, processed, digested with trypsin and then subjected to MALDI-TOF MS (panel B, image 3). In the protein sequence of the GPI-PLC the peptides detected are underlined. The symbol * above a lysine (k) represents either an unblocked lysine within the detected peptide or an unblocked lysine immediately preceeding the detected peptide, after which trypsin has successfully cleaved. The symbol † above a lysine (k) represents a position within the sequence that has not yet been determined by MALDI-TOF MS to be un-derivatized.
Figure 2.
The GPI-PLC can be surface radio-iodinated in bloodstream form trypanosomes.
Autoradiographs (15 day exposure) of the immunoprecipitates of the GPI-PLC in non-penetrating surface radio-iodinated GPI-PLC+/+ bloodstream forms (4.2×107 cell equivalents per track) of T.brucei using either 95 µg of purified non-immune IgG with GPI-PLC+/+ (track 1), or using 77.8 µg of anti-GPI-PLC IgG to compare GPI-PLC−/− (track 2) and GPI-PLC+/+ (track 3) bloodstream forms of T. brucei.
Figure 3.
Confocal images of the GPI-PLC in GPI-PLC+/+, GPI-PLC−/−and procyclic form trypanosomes and the YFP-tagged GPI-PLC in recombinant bloodstream form cells.
Each of the panels, A–E, contains a merged image of the nucleus and kinetoplast, together with the GPI-PLC probed with anti-GPI-PLC antibody (magenta) and the phase image; (panel A), wild-type GPI-PLC+/+ bloodstream form trypanosome; (panel B), a GPI-PLC−/− null mutant bloodstream form trypanosome; (panel C), a wild-type procyclic form trypanosome; (panel D), a low power view of wild-type GPI-PLC+/+ bloodstream form trypanosomes and (panel E), a low power view of GPI-PLC−/− null mutant bloodstream form trypanosomes. The phase image (panel F) of S-42 stumpy bloodstream forms shows the typical morphology of these forms in this strain, while the fluorescence image (panel G) shows the location of the GPI-PLC (magenta) and the merge of these two images shows their spatial relationship (panel H). The location of YFP-tagged GPI-PLC is shown in the composite image of six bloodstream forms in panel I and the arrowheads point to the endo-membrane-bound spaces containing a small amount of GPI-PLC. Bars, 5 µm.
Figure 4.
Location of the GPI-PLC compared to the location of a series of markers in T. brucei.
Each panel shows the comparative locations of the GPI-PLC (magenta) and a marker; panel A, tubulin marker (cyan); panel B, paraflagellar rod marker (yellow); panel C, FAZ1 protein marker (orange); panel D, non-penetrating membrane protein reactive dye marker (green). (1), merge of the differential interference contrast image (DIC) and the Hoechst image of the nucleus and kinetoplast; (2), GPI-PLC; (3), marker; (4), merge of all images in the panel except the DIC image and (5) is the merge of all images. Arrowheads mark the location of a small amount of the GPI-PLC in a second endomembrane-bound compartment (Panel A-2). Upper large insets in panels B (4) and C (4) are the adjacent regions in panels B (4) and C (4) respectively with the saturation and brightness increased. Lower small insets in panels C (2), C (3) and C (4) are the adjacent regions in these panels with the contrast increased in the C (2) inset for clarity and contain small arrows marking the end of the structure containing both the GPI-PLC and the FAZ1 proteins. Upper arrows mark the end of the GPI-PLC linear array; lower arrows mark the end of the PFR in panel B (4) and the FAZ1 protein array in panel C (4). Arrowhead in panel C (4) indicates a region where separation of the FAZ1 protein and the GPI-PLC is particularly clearly demonstrated. Bars, 5 µm.
Figure 5.
Effect of temperature and detergent on the detection of the VSG, tubulin, ISG-70 and GPI-PLC.
Panel A shows the detection of VSG, tubulin, ISG-70 and GPI-PLC in wild type bloodstream form trypanosomes fixed at 0°C in the presence of triton X-100. Panel B shows the detection of VSG, tubulin, ISG-70 and the GPI-PLC in wild type bloodstream form trypanosomes fixed at 0°C in the absence of triton X-100. Panel C shows the detection of VSG, tubulin, ISG-70 and GPI-PLC in wild type bloodstream form trypanosomes fixed at 37°C in the presence of Tx-100. Panel D shows the detection of VSG, tubulin, ISG-70 and GPI-PLC in wild type bloodstream form trypanosomes fixed at 37°C in the absence of triton X-100. The inset in each panel is the Differential Interference contrast (DIC) image. Bars, 5 µm.
Figure 6.
The location of GPI-PLC compared to a membrane marker and the FAZ1 protein in cells with a partially detached flagellum.
Panel A shows the comparative location of the FAZ1 protein (orange) and a non-penetrating membrane protein reactive dye (green); (1), merge of the DIC image and image of the nucleus and kinetoplast; (2), FAZ1 protein; (3), membrane protein; (4), merge of all images in the panel except the DIC image and (5) is the merge of all images. Panel B shows the comparative location of the GPI-PLC (magenta) and a non-penetrating membrane protein reactive dye (green); (1), merge of DIC image and image of the nucleus and kinetoplast; (2), GPI-PLC; (3), membrane protein; (4), merge of all images in the panel except the DIC image and (5) is the merge of all images. The brightness of panel A image 4 and panel B, image 4 have been uniformly increased to the same extent for ease of viewing. Bars, 5 µm.
Figure 7.
Location of the GPI-PLC in intact cells before and after release of the VSG.
Panel A shows a cell in a fully energized trypanosome incubated for 45 min at 37°C in the presence of glucose (10 mM). Panel B, shows a cell incubated for 45 min at 37°C without glucose but with 2-deoxyglucose (10 mM) added. In each case panel (1) is a merge of the DIC (gray) and Hoechst stained nucleus and kinetoplast (blue) images, panel (2) is the GPI-PLC image (magenta) and panel (3) is a merge of all images. Panel C shows the time course of the release of the VSG from bloodstream form trypanosomes in the absence of glucose but in the presence of 2-deoxyglucose measured as absorbance of the supernatant of cells at 280 nm. Panel D, image 1 shows the Coomassie blue stained proteins after SDS-PAGE of bloodstream form trypanosomes (MITat 1.1) incubated for 60 min in the presence of glucose (track a) and in the absence of glucose but with 2-deoxyglucose added (track b). Panel D, image 2 shows an equivalent western blot. Bars, 5 µm.
Figure 8.
Scheme 1. Cartoons showing the arrangement of VSG dimers shielding the GPI-PLC at 37°C and allowing access to the GPI-PLC at 0°C as well as the cross-sectional relationships between the GPI-PLC and other surrounding structures.
Panel A. Cartoon of the arrangement of VSG dimers (red & blue) on the outer leaflet of the plasma membrane of bloodstream form trypanosomes after mild cross-linking (black bars) of proteins by brief fixation (10 min) at either 37°C or 0°C. Subsequent access to the VSG is shown by the position of attached IgG anti-VSG (grey, mauve and turquoise) and access to a partially buried protein, such as the GPI-PLC (mauve), is shown by the position of attached or excluded IgG anti-GPI-PLC (yellow, green and grey). Panel B. Cartoon of a cross section through the cell body and flagellar compartment showing the relationships between the GPI-PLC and the flagellar attachment zone together with associated structures, including the ACPSMT, axonemal central pair single mictotubule; APMTD, axonemal peripheral microtubule doublet; DMT, diminished microtubule [29]; ER, loop of endoplasmic reticulum contacting the four constant microtubules within the flagellar attachment zone; FAZDB, flagellar attachment zone dense body; GPI-PLC (magenta), the glycosylphospatidyl-inositol phospholipase C; N, nucleus covered by inner and outer nuclear membranes; PMT, pellicular microtubule; PFR, paraflagellar rod; SCAC, surface coat covering the plasma membrane of the flagellar compartment; SCBC, surface coat covering the plasma membrane of the cell body compartment; TMF, one of the transmembrane fibrils connecting the paraflagellar rod to the flagellar attachment zone dense body.