Flagellar Pocket Membrane Research Articles

A differential role for actin during the life cycle of Trypanosoma brucei.

Actin is expressed at similar levels but in different locations in bloodstream and procyclic forms of Trypanosoma brucei. In bloodstream forms actin colocalizes with the highly polarized endocytic pathway, whereas in procyclic forms it is distributed throughout the cell. RNA interference demonstrated that in bloodstream forms, actin is an essential protein. Depletion of actin resulted in a rapid arrest of cell division, termination of vesicular traffic from the flagellar pocket membrane leading to gross enlargement of the pocket, loss of endocytic activity and eventually cell death. These results indicate that actin is required for the formation of coated vesicles from the flagellar pocket membrane, which is the first step in the endocytic pathway. Although loss of actin in procyclic cells did not affect growth, the trans region of the Golgi became distorted and enlarged and appeared to give rise to a heterogeneous population of vesicles. However, the flagellar pocket was not affected. These findings suggest that trypanosomes have different functional requirements for actin during the bloodstream and procyclic phases of the life cycle.

Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers.

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endo- and exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50-60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11- as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.

Elemental composition of acidocalcisomes of Trypanosoma cruzi bloodstream trypomastigote forms.

The elemental composition of acidocalcisomes present in Trypanosoma cruzi bloodstream trypomastigotes was analyzed by X-ray microanalysis. Acidocalcisomes appear as electron-dense vesicles dispersed throughout the cytoplasm. X-ray microanalysis of whole cells adhered to Formvar-coated grids showed that they contained C, O, Na, Mg, P, S, Ca and Zn, although in some acidocalcisomes iron was also detected. However, in ultra-thin sections of parasites processed according to routine procedures, most elements were lost. Living parasites were incubated with gold-labeled holo-transferrin to test if the iron was taken up by receptor-mediated endocytosis. After incubation at 4 degrees C, labeling was observed only in the flagellar pocket membrane, showing that receptors to this protein are already present in bloodstream trypomastigotes. In parasites incubated at 28 degrees C no intracellular tracer was observed, despite the labeling in the flagellar pocket membrane. Our data show that some acidocalcisomes of T. cruzi bloodstream trypomastigotes contain iron and that this element was not taken up by receptor-mediated endocytosis.

Sequence requirements for trafficking of the CRAM transmembrane protein to the flagellar pocket of African trypanosomes.

CRAM is a cysteine-rich acidic transmembrane protein, highly expressed in the procyclic form of Trypanosoma brucei. Cell surface expression of CRAM is restricted to the flagellar pocket of trypanosomes, the only place where receptor mediated endocytosis takes place in the parasite. CRAM can function as a receptor and was hypothesized to be a lipoprotein receptor of trypanosomes. We study mechanisms involved in the presentation and routing of CRAM to the flagellar pocket of insect- and bloodstream-form trypanosomes. By deletional mutagenesis, we found that deleting up to four amino acids from the C terminus of CRAM did not affect the localization of CRAM at the flagellar pocket. Shortening the CRAM protein by 8 and 19 amino acids from the C terminus resulted in the distribution of the CRAM protein in the endoplasmic reticulum (ER) (the CRAM protein is no longer uniquely sequestered at the flagellar pocket). This result indicates that the truncation of the CRAM C terminus affected the transport efficiency of CRAM from the ER to the flagellar pocket. However, when CRAM was truncated between 29 and 40 amino acids from the C terminus, CRAM was not only distributed in the ER but also located to the flagellar pocket and spread to the cell surface and the flagellum. Replacing the CRAM transmembrane domain with the invariant surface glycoprotein 65-derived transmembrane region did not affect the flagellar pocket location of CRAM. These results indicate that the CRAM cytoplasmic extension may exhibit two functional domains: one domain near the C terminus is important for efficient export of CRAM from the ER, while the second domain is of importance for confining CRAM to the flagellar pocket membrane.

A novel protein targeting domain directs proteins to the anterior cytoplasmic face of the flagellar pocket in African trypanosomes.

The flagellar pocket of African trypanosomes is a critical sorting station for protein and membrane trafficking, and is considered to be an Achilles' heel of this deadly pathogen. Although several proteins, including receptors for host-derived growth factors, are targeted specifically to the flagellar pocket, the signals responsible for this restricted subcellular localization are entirely unknown. Using T lymphocyte triggering factor-green fluorescent protein (TLTF(1)-GFP) fusion proteins, we demonstrate that an internal 144 amino acid domain of TLTF from Trypanosoma brucei is sufficient for directing GFP to the cytoplasmic side of the anterior flagellar pocket. Immuno-gold electron microscopy reveals that the TLTF-GFP fusion protein is located in an electron dense structure that immediately abuts the anterior flagellar pocket membrane. The amino acid sequence of the TLTF targeting domain does not resemble previously characterized protein trafficking signals, and random mutagenesis reveals that flagellar pocket targeting is conferred by a structural motif, rather than a short, contiguous array of amino acids. The aberrant sorting of two mutant proteins into the flagellum, and the targeting of a related human protein to the plus end of the trypanosome's cytoskeletal microtubules, lead us to suggest that flagellar pocket targeting involves interactions with the trypanosome cytoskeleton. The finding that TLTF-GFP is restricted to the anterior, cytoplasmic face of the flagellar pocket membrane, suggests that there is structural heterogeneity in the membrane of this organelle.

The Cytostome of Trypanosoma cruzi Epimastigotes Is Associated with the Flagellar Complex

Okuda, K., Esteva, M., Segura, E. L., and Bijovsky, A. T. 1999. The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex. Experimental Parasitology92, 223–231. Proliferative forms of Trypanosoma cruzi, amastigotes and epimastigotes, have a cytostome, a specialized structure formed by an invagination of the flagellar pocket's membrane surrounded by microtubules and frequently followed by a row of vesicles. All this assemblage penetrates deeply into the cytoplasm overpassing the nucleus. This structure, together with the flagellar pocket, appears to play an important role in the nutrition of the parasite. We demonstrated that the monoclonal antibody 2C4, made-up against isolated flagellar complex of T. cruzi epimastigotes, recognizes a protein doublet of 76 and 87 kDa in total epimastigotes homogenate. The 76-kDa polypeptide is enriched in the detergent-soluble fraction whereas the 87-kDa polypeptide is highly represented in the insoluble fractions and the purified flagella. Immuno-fluorescence assays show the antigen as a small spot at the flagellar pocket region. Immunogold labeling of ultrathin sections of epimastigote forms reveals gold particles at the opening of flagellar pocket, concentrated in the cytostome region. Immunocytochemistry of epimastigote whole-mount cytoskeletons reveals the labeling on an array of three to four microtubules that appears attached to flagellum, running in the direction of the nucleus. Ultrastructural observations have shown that the posterior region of isolated flagella, corresponding to the level of the flagellar pocket, possesses a microtubular structure compatible with that from the cytostome. The relationship between the cytostome, an endocytic organelle, and the flagellum is here described for the first time.

Developmentally Regulated Sensitivity of Trypanosoma brucei brucei to the Cytotoxic Effects of Human High-Density Lipoprotein

The bloodstream developmental stages of the protozoan parasite Trypanosoma brucei brucei are lysed by normal human serum. The cytotoxic factor is a minor sub-class of human high-density lipoprotein termed trypanosome lyric factor (TLF). T. b. brucei rapidly develops resistance to TLF when incubated at 26°C under conditions which allow differentiation to the procyclic, insect midgut developmental stage. This in vitro differentiation system allowed us to correlate loss of TLF sensitivity with other parameters of differentiation to the procyclic form. The onset of resistance to TLF occurs within 2 hr after shifting bloodstream forms to differentiation conditions. TLF resistance is correlated with a rapid but transient decrease in protein synthesis by the parasite, is acquired prior to cell division at 26°C, and precedes the loss of variant surface glycoprotein. Tn addition, we found binding and uptake of TLF by established procyclic trypanosomes was reduced approximately fivefold relative to bloodstream trypanosomes and the TLF binding observed in procyclics was nonspecific. No TLF was bound to the procyclic flagellar pocket membrane and the procyclics failed to endocytose any of the surface-bound TLF. In contrast, bloodstream forms bind TLF via a flagellar pocket-localized protein and bound TLF is taken up;by endocytosis. These findings suggest that resistance of procyclic T. b. brucei to TLF-mediated lysis is due to a reduction in the endocytosis of TLF by this developmental stage of the parasite.

A novel method for mean cell volume estimation

SummaryA novel method is described for the estimation of the mean cell volume of cell populations grown in suspension. The cells are filtered onto a nitrocellulose filter to form a cylindrical pellet which is embedded in epoxy resin. Using estimates of pellet height and radius, the number of cells in the pellet and of the volume density of the cells in the pellet, it is possible to produce an unbiased estimate of the mean cell volume. This method is compared, using cell suspensions of the blood parasite Trypanosoma brucei, with mean cell volume estimation using a Coulter channellizer. A Coulter channellizer was also used to compare the mean cell volume of living trypanosomes with that of aldehyde‐fixed populations, and the values obtained were compared with those obtained using the new method. The estimated mean cell volume of a T. brucei clone was used to derive values from volume densities obtained by point and intersection counts for the absolute volumes of the flagellar pocket, the nucleus, and endocytic organelles containing internalized horseradish peroxidase and transferrin‐gold after 30‐min incubations at 310 K. Estimated values for the surface area of the flagellar pocket and the surface area of a cell were also obtained. From known data on the total amount of variant surface glycoprotein molecules per cell and the known packing density of membrane proteins, it was estimated that approximately 80% of the molecules must reside in intracellular compartments. It was estimated that the equivalent of 5% of the surface membrane may be internalized per minute, an amount which is almost the size of the entire flagellar pocket membrane.

Failure of immunization with trypanosome endocytotic vesicle membrane proteins to provide nonvariant immunoprotection against Trypanosoma brucei.

Purified trypanosome endocytotic vesicles were subjected to Triton X-114 phase separation to obtain a fraction enriched in putative parasite receptors for adsorptive endocytosis. Rabbits immunized with this material produced antibodies that recognized many parasite proteins, including nonvarying epitopes on the parasite's endocytotic surface, the flagellar pocket membrane, as well as on membranes of endosomes and lysosome-like structures. These antibodies were unable to stimulate in vitro complement-mediated lysis of trypanosomes, and in an in vitro test of parasite growth inhibition they actually marginally enhanced parasite proliferation. No effect was observed on the parasite prepatent period or parasitemia in mice injected with antibody purified from the rabbit antisera, but their survival with the infection was significantly shortened. Finally, little difference was detected in parasitologic or hematologic parameters between immunized and control rabbits upon challenge with T. brucei infection.

Identification of membrane-associated proteins in Trypanosoma brucei encoding an internal, EARLRAEE amino acid repeat.

Differential screening of expression libraries with nonimmune and day 4-6 immune serum from naive rats infected with the protozoan Trypanosoma lewisi was used to identify potential cell surface protein coding genes. Several T. lewisi cDNAs that resulted were partially characterized and used to clone the homologues from Trypanosoma brucei. The nucleotide sequence of the cDNAs encoding the Tb-29 genes of the parasitic protozoan T. brucei obtained by this method (3557 nucleotides for Tb-291 and 8729 nucleotides for Tb-292) encoded predicted open reading frames of 1070 and 2550 amino acids, respectively. The Tb-29 proteins encoded a large domain with an octapeptide (EARLRAEE) repeat (79 repeats in Tb-291 and 60 in Tb-292), which shared significant similarity with the octapeptide repeat of the S-antigen of isolate NF7 of Plasmodium falciparum. The predicted amino acid sequence of the Tb-292 protein encoded potential transmembrane domains (eight in total). Indirect immunofluorescence using confocal image analysis and immunoelectron microscopy located the EARLRAEE proteins to a membranous network. The Tb-29 proteins were most abundantly distributed to the area surrounding the nucleus, the region between the nucleus and the flagellar pocket, and the region immediately underneath the flagellar pocket membrane. The subcellular distribution of Tb-29 proteins suggests that these proteins may provide a constituent associated with the cell's vesicular transport system.

Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi.

Epimastigote forms of the pathogenic parasite Trypanosoma cruzi were used to study the endocytic process in a protozoon. These elongated unicellular organisms are highly polarized cells: endocytosis occurs only at the anterior region through the cytostome and the flagellar pocket membrane, areas of the plasma membrane where the cell cytoskeleton, formed by sub-pellicular microtubules, is absent. When the cells were incubated at 4 degrees C or 28 degrees C with gold-labeled transferrin, fixed and processed for routine transmission electron microscopy our observations show that this ligand initially binds to the cytosome and the membrane lining the flagellar pocket and is subsequently ingested through a clathrin-independent receptor-mediated endocytotic process, with formation of uncoated pits and vesicles. Ingested complexes are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the posterior end of the cell. Immunocytochemical data from Lowicryl-embedded cells demonstrated that the reservosomes are acidic compartments (pH 6.0, as shown using DAMP as a pH probe) with no acid phosphatase or typical lysosome-associated membrane proteins (LAMP 1, LAMP 2 and lgp 120), but rich in cysteine proteinase. These data suggest that the reservosome is a pre-lysosomal compartment. Since cysteine proteinase of T. cruzi contains no phosphorylated mannose residues and the cation-independent mannose 6-phosphate receptor could not be immunocytochemically detected in the reservosomes, it is possible that lysosomal enzymes in the epimastigote forms of T. cruzi are targeted to compartments related to the endocytic pathway through a mechanism different from that which occurs in other eukaryotic cells.

Freeze‐Fracture Study of the Bloodstream Form of Trypanosoma brucei gambiense

The ultrastructure of Trypanosoma brucei gambiense was investigated by the freeze-fracture method. Three different regions of the continuous plasma membrane; cell body proper, flagellar pocket, and flagellum were compared in density and distribution of the intramembranous particles (IMP's). The IMP-density was highest in the flagellar pocket membrane and lowest in flagellum. Intra membranous particles of the cell body membrane were distributed uniformly on both the protoplasmic (P) and exoplasmic (E) faces. On the P face of the flagellar membrane, a single row of IMP-clusters was seen along the juncture of the flagellum to the cell body. Since the spacing of the IMP-clusters was almost equal to the spacing of the paired rivet structures observed in thin section, these clusters likely are related to the junction of flagellum and cell body. At the neck of the flagellar pocket, several linear arrays of IMP's were found on the P face of the flagellar membrane, while on the E face rows of depressions were seen. At the flagellar base, the clusters of IMP's were only seen on the P face. On the flagellar pocket membrane, particle-rich depressions and linear particle arrays were also found on the P face, while on the E face such special particle arrangements were not recognized. These particle-rich depressions may correspond to the sites of pinocytosis of the bloodstream forms which have been demonstrated in thin sections.

Receptors for the host low density lipoproteins on the hemoflagellate Trypanosoma brucei: purification and involvement in the growth of the parasite.

The slender bloodstream form of Trypanosoma brucei shows receptor-mediated endocytosis of low density lipoprotein (LDL) particles of its hosts. We have purified the LDL receptor of this species nearly to homogeneity (about 1000-fold purification) and obtained monospecific polyclonal antibodies against it. As analyzed by NaDodSO4/polyacrylamide gel electrophoresis, the purified receptor consists of a single subunit, with an apparent molecular mass of 86 kDa. Its isoelectric point is 5.9. On the average, each cell exposes 52,000 copies of low-affinity receptors (Kd of 250 nM) and 1800 copies of high-affinity receptors (Kd of 5.7 nM). According to indirect en bloc immunolabeling of fixed parasites, the receptor appears to be localized to the flagellar pocket membrane and the flagellar membrane and to be completely absent from the rest of the pericellular membrane. LDL is required for optimal growth of the trypanosome in vitro: cell growth can be inhibited either by removal of LDL from the culture medium or by antibodies against the purified LDL receptors. In both cases, growth is restored by the addition of excess LDL.

A flagellar pocket membrane fraction from Trypanosoma brucei rhodesiense: immunogold localization and nonvariant immunoprotection

In contrast to the abundance of detailed information on variant-specific surface coat antigens of African trypanosomes, data on possible common or nonvariant antigens within these protozoa are surprisingly limited. In this study, the cellular localization and protective potential of a previously characterized flagellar pocket membrane (FPM) fraction were determined. Immunogold staining of live trypanosome suspensions at 0 to 4 degrees C by using anti-FPM hyperimmune serum raised in rabbits as the primary antibody revealed specific staining of the parasite surface at the emergence of the flagellum from the flagellar pocket. The same specificity of immunogold localization was obtained for each of three distinct variable antigenic types (VATs) of a serodeme of Trypanosoma brucei rhodesiense Wellcome strain. Products of translated mRNA preparations from each of the VATs were precipitated by the FPM antiserum and revealed identical banding patterns when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Immunization of mice with FPM fraction protected them against infection by two of the VATs; the third VAT was afforded poor protection. This is the first demonstration of the combined cellular localization, nonvariant nature, and protective potential of a membrane fraction from African trypanosomes.

Trypanosoma rhodesiense: Antigenicity and immunogenicity of flagellar pocket membrane components

A low density membrane fraction, isolated from the bloodstream stage of Trypanosoma rhodesiense and enriched in flagellar pocket membrane, was characterized with regard to antigenicity using antibodies raised against purified flagellar pocket membrane. Mild trypsinolysis of flagellar pocket membrane released two small peptides ( M r = 13–16 × 10 3) separated by chromatofocusing (p I = 6.8 and 5.8) that were antigenic as monitored by fused rocket immunoelectrophoresis. Both of these antigenic peptides were enriched in relative fluorescence when flagellar pocket membrane was prepared from surface labeled (fluorescamine-β-cyclodextrin) trypanosomes, indicating that cleaved peptides were on the external (luminal) side of the flagellar pocket membrane. More extensive release of fluorescamine labeled flagellar pocket membrane components was affected using mild detergent treatment (0.15% Zwittergent 3–12 0.4% Triton X-100), crossed immunoelectrophoresis separating two prominent and a third much fainter antigen. Release of the more anodic of the two prominent antigens was more pronounced after incubation of flagellar pocket membrane with either porcine pancreas phospholipase A 2 or umbilical cord sphingomyelinase. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent electroblotting to nitrocellulose also revealed two principal flagellar pocket membrane antigens ( M r ≈ 60 and 66 × 10 3), the latter showing greater release after exposure to sphingomyelinase or phospholipase, compared to mild detergent or 50 m M acetate, pH 5.0. Both antigens were glycoprotein as judged by electroblotting and the use of concanavalin A conjugated horseradish peroxidase as probe. Neither flagellar pocket membrane antigen was found to react with monoclonal antibodies prepared against T. rhodesiense variable surface antigen. The use of flagellar pocket membrane in the presence of Freund's complete adjuvant was found to protect mice against challenge infections with either the CP344 clone or uncloned CT Wellcome isolate of T. rhodesiense.

"Benadryl" in Treatment of Oxyuriasis

<h3>Abstract</h3> Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. African trypanosome pentamidine and melarsoprol sensitivity is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation or via binding to TbAQP2, with subsequent endocytosis, but trafficking and regulation of TbAQPs is uncharacterised. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation also leads to impaired oligomerisation, mislocalisation, and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and contributes towards emergence of drug resistance.