WIPI49 is a member of a previously undescribed family of WD40-repeat proteins that we demonstrate binds 3-phosphorylated phosphoinositides. Immunofluorescent imaging indicates that WIPI49 is localized to both trans-Golgi and endosomal membranes, organelles between which it traffics in a microtubule-dependent manner. Live cell imaging establishes that WIPI49 traffics through the same set of endosomal membranes as that followed by the mannose-6-phosphate receptor (MPR), and consistent with this, WIPI49 is enriched in clathrin-coated vesicles. Ectopic expression of wild-type WIPI49 disrupts the proper functioning of this MPR pathway, whereas expression of a double point mutant (R221,222AWIPI49) unable to bind phosphoinositides does not disrupt this pathway. Finally, suppression of WIPI49 expression through RNAi, demonstrates that its presence is required for normal endosomal organization and distribution of the CI-MPR. We conclude that WIPI49 is a novel regulatory component of the endosomal and MPR pathway and that this role is dependent upon the PI-binding properties of its WD40 domain.

The mammalian-infective bloodstream form of Trypanosoma brucei possesses a highly active endocytotic system. Evasion of the host immune response by T. brucei is dependent on antigenic variation of VSG (variant surface glycoprotein), but additional mechanisms for removal of surface-bound antibody also operate. Four Rab proteins, Tb (trypanosomal) RAB4, 5A, 5B and 11 are located to the endosomal system; TbRAB5A and TbRAB11 co-localize with internalized anti-VSG antibody and transferrin. A live cell assay was used to record a single cycle of endocytosis of anti-VSG IgG and transferrin, their subsequent degradation within the endosomal system and exocytosis of the products. TbRAB5A and TbRAB11 were involved in the overall process of endocytosis, degradation and exocytosis, whereas TbRAB5B and TbRAB4 were not implicated. The kinetics of anti-VSG IgG and transferrin recycling depend on the nucleotide state of TbRAB5A and TbRAB11. These data, together with previous work, suggest that IgG and transferrin initially enter a TbRAB5A sorting endosome and are most probably recycled subsequently via a TbRAB11-dependent step. Analysis of the recycled IgG and transferrin demonstrated extensive degradation of these recycled proteins. Degradation of transferrin was enhanced in cells expressing increased amounts of TbRAB5A or TbRAB11 with a Ser-->Asn mutation, but was decreased when active TbRAB11 was overexpressed. The extent of degradation of anti-VSG IgG was found to be unaffected by mutant Rab protein expression. The presence of an efficient mechanism for the removal of IgG bound to the external surface of T. brucei and its subsequent proteolysis within the recycling system suggests a role for this pathway in immune evasion.

Eukaryotic flagella and cilia are among the most ancient of cellular organelles, and their basic architecture is conserved from protozoa to vertebrates (28). The core structure of the flagellar/ciliar axoneme comprises a membrane-bound cylinder of nine microtubule doublets plus two central singlet microtubules. Formation of the axoneme is organized by basal bodies, cylindrical organelles with walls composed of nine triplet microtubules that serve as templates for the assembly of doublet microtubules. Basal bodies and centrioles are similar in structure, and these organelles are often interchangeable in many organisms (4). The functions of flagella and cilia include roles in development, metazoan body polarity, and cell division, while many diseases involve a defect in the function of flagellar components (17).The protozoan Trypanosoma brucei is an ideal system in which to study microtubule-mediated events, because, in addition to the flagellum, the cell is highly polarized as a consequence of an ordered array of subpellicular microtubules (8,11). The trypanosome has a single flagellum that emerges from the flagellar pocket, an invagination of the plasma membrane at the posterior end of the cell (9, 21). The axoneme of kinetoplastid protozoa is attached to a lattice-like structure of a similar diameter, the PFR, extending along most of the axoneme (9). The flagellum is attached to the cell body throughout most of its length by the FAZ, which is composed of an electron-dense filament plus four cytoplasmic microtubules that originate from the basal body region (9).During cell division the trypanosome must replicate and separate several single-copy organelles present in G 1 cells, i.e., nucleus, kinetoplast, mitochondrion, basal body, and flagellum (36, 44). Ultrastructural studies have described a number of markers of cell cycle position and elucidated a number of discrete cell cycle phases (36). The first morphological event of the T. brucei cell cycle is the maturation and duplication of the basal body, which is followed by kinetoplast S phase and then nuclear S phase (36,44). During flagellum morphogenesis, the new axoneme is assembled from the recently matured basal body. New probasal bodies are formed, and the elongation and emergence of the flagellum from the flagellar pocket is fol-* Corresponding author. Present address:

No abstract

Essentially all macromolecular communication between Trypanosoma brucei and its host is confined to vesicular trafficking events occurring at or around the flagellar pocket. The vertebrate stage bloodstream form trypomastigote exhibits an extremely high rate of endocytosis required for nutrient uptake and probably also evasion of the host immune system. However, the rate of endocytosis is very low in the procyclic vector parasite, indicating that endocytosis is subject to a marked level of developmental regulation. Previous ultrastructural studies and crude biochemical fractionations have indicated the presence of coated pits and vesicles that are analogous to clathrin coats in the bloodstream form, but not in the procyclic. However, a definitive description of the components of this coat and its molecular function in T. brucei has remained elusive. We describe the molecular cloning and initial characterisation of components of the T. brucei endocytic coats: clathrin heavy chain (TbCLH) and a β-adaptin (TbAPβ1). TbCLH is markedly upregulated in the bloodstream form compared with the procyclic, whereas TbAPβ1 is subject to more limited developmental regulation. We generated antisera against both proteins and show that the clathrin coat is tightly associated with the flagellar pocket in both major life stages. However, in bloodstream parasites TbCLH is also extensively distributed throughout the posterior end of the cell on numerous large vesicular and tubular structures. By cryoimmuno EM, clathrin is localised to collecting tubules at the flagellar pocket and is also associated with the trans-Golgi network. These EM data confirm that the electron dense coats reported on trypanosome vesicles and tubules contain clathrin. The TbAPβ1 exhibits an atypical distribution relative to previously characterised adaptins, associating not only with the trans-Golgi but also with other tubular-vesicular elements. Localisation of TbAPβ1 is also subject to developmental regulation. These data describe major endocytic coat proteins in T. brucei for the first time, and indicate stage-specific expression of the clathrin heavy chain. Modulation of clathrin expression is likely to be an important factor in the developmental regulation of endocytosis and recycling in the African trypanosome.

Endocytosis in the parasitic protozoan Trypanosoma brucei, a deeply divergent eukaryote, is implicated as important in both general cellular function and virulence, and is strongly developmentally regulated. We report the characterisation of a previously undefined endosomal compartment in T. brucei based on identification of a new trypanosome gene (TbRAB11) homologous to Rab11/Ypt31. Northern and western analyses indicated that TbRAB11 expression was significantly upregulated in the bloodstream stage of the parasite, the first trypanosome Rab to be identified with a developmentally regulated expression profile. In procyclic form parasites TbRAB11 localised to a compartment positioned close to the basal body, similar to mammalian Rab11. By contrast, in bloodstream form parasites, TbRAB11-containing structures were more extensive and the TbRAB11 compartment extended towards the posterior face of the nucleus, was more elaborate and was not always adjacent to the basal body. Colocalisation studies by light and confocal microscopy demonstrated that TbRAB11 was located on a compartment that did not correspond to other established trypanosomal organelles or markers. Using concanavalin A internalisation and temperature block procedures, TbRAB11 was observed on endomembranes anterior to the flagellar pocket that are juxtaposed to the collecting tubules. TbRAB11 colocalised with the trypanosomal transferrin receptor and internalised antivariant surface glycoprotein. Further, we show that the collecting tubules contain TbRAB5A, suggesting that they are the trypanosomatid early endosome. Hence, TbRAB11 is present on endosomal structures that contain recycling cargo molecules and is under developmental regulation, suggesting a role in stage-dependent endocytic processes.

The protozoan parasite Toxopkzsma gondii is an important cause of fetal mortality in sheep. Infection occurs when sheep ingest feed or water contaminated with oocysts excreted by the cat. A primary infection in pregnant sheep can establish a placental and fetal infection which may result in fetal death and resorption, abortion or stillbirth.We applied the polymerase chain reaction (PCR), using SAG1 and B1 gene primers, to detect the presence of Toxopkzsma gondii in placental tissues and aborted or stillborn lamb tissues from a farm population. 97 lambings (total of 158 lambs) were screened by PCR with 36.1% showing positive amplification. Of the 21 problem pregnancies 57.1% were positive by PCR. The high prevalence of Toxoplasma gondii observed in stillborn lambs indicates the importance of congenital transmission in sheep. Furthermore, the high prevalance in successful lambings suggests that pathology is not always associated with infection. This study demonstrates the relevance of direct analysis of Toxoplasma gondii infection in tissue samples in Toxoplusma population ecology. A 'naturally' yellow fluorescent variant surface glycoprotein (VSG) from Trypanosoma bruceiWe have identified a wild-type bloodstream form variant of Trypanosoma h c e i that exhibits a yellow fluorescence of the cell surface, including the flagellar pocket membrane. By cloning of the active VSG gene from the 'yellow' variant and expression in a non-fluorescent trypanosome line we confirmed that the fluorescence is VSG-mediated. The fluorescence emission is exceptionaIly high, fades slowly and recovers within one minute after photobleaching. Interestingly, the 'yellow' VSG is not a classic autofluorescent protein, but requires short wave length excitation energy and binding of a compound from the cell culture medium. This feature allows us to switch the surface fluorescence on and off by a simple medium exchange. We have exploited the advantages of the intrinsic, inducible VSG surface fluorescence for the visualization and analysis of vesicular trafficking close to the flagellar pocket in living trypanosomes. For that purpose we have generatcd transgenic 'yellow' trypanosomes expressing both, a procyclin:DsRed chimera and a G F P fusion protein, that illuminates the secretory pathway. An analysis of this cell line by multi-channel 3d-microscopy is presented. Flagellar targeting of proteins in Trypanosono bruceiKlaus Ersfeld and Keith Gull School of Biological Sciences, 2.205 Stopford Building, University of Manchester. Oxford Road, Manchester M13 9PT, UK.Although the flagellum of eukaryotes is a highly distinct organelle contain approximately 250 different proteins, very little is know about the mechanisms which target specific proteins to the flagellar compartment. In trypanosomes and some related protists an additional level of complexity of the flagellar protein complement is given by the existence of the paraflagellar rod (PFR), a highly ordered structure running alongside the axoneme. The PFR is composed of two well characterised ma...