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 flagllum 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.     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Leishmania mexicana: distribution of intramembranous particles and filipin sterol complexes in amastigotes and promastigotes

The density and distribution of intramembranous particles was analyzed in freeze fracture replicas of the plasma membrane of amastigotes, and infective as well as noninfective promastigotes of Leishmania mexicana amazonensis. The density of intramembranous particles on both protoplasmic and extracellular faces was higher in infective than in noninfective promastigotes and it was lower in amastigotes than in promastigotes. Amastigotes purified immediately after tissue homogenization were surrounded by a membrane which corresponded to the membrane which lined the endocytic vacuoles where the parasites were located within the tissue macrophages. Aggregation of the particles was seen in the flagellar membrane at the point of emergence of the flagellum from the flagellar pocket. Differences in the organization of the particles were seen in the membrane which lined the flagellar pocket of amastigotes and promastigotes. The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the plasma membrane of L. m. amazonensis. The effect of filipin in the parasite's structure was analyzed by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze fracture replicas. Filipin sterol complexes were distributed throughout the membrane which lined the cell body, the flagellar pocket, and the flagellum. No filipin sterol complexes were seen in the cell body-flagellar adhesion zone. The density of filipin sterol complexes was lower in the membrane lining the flagellum than in that lining the cell body of promastigotes.     

Tritrichomonas foetus: Fine Structure of Freeze-Fractured Membranes1

ABSTRACT. Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9–12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/μm² and 468/μm² respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per μm² in the nuclear. membrane.     

Fine structure of Blastocrithidia culicis as seen in thin sections and freeze-fracture replicas

The fine structure of epimastigotes of Blastocrithidia culicis was studied by transmission electron microscopy of thin sections and freeze-fracture replicas. This parasite presents a well developed endoplasmic reticulum and Golgi complex systems. Differences in the density and organization of the intramembranous particles were observed between the membranes which enclose the cell body and the flagellum. Ridge-like elevations, visualized in freeze-fracture replicas, were observed in sites where the mitochondrial branches touched the plasma membrane. A special array of membrane particles was observed on both faces of the flagellar and the cell body membranes at the region where the flagellum adheres to the cell body. It appeared as strands made of two rows of membrane particles. Filipin-treated cells were used for the localization of membrane sterols in freeze-fracture replicas. The number of filipin-sterol complexes varied from cell to cell. In some cells, rows of filipin-sterol complexes were seen. No complexes were observed in the region of the attachment of the flagellum to the cell body.     

Trypanosoma brucei rhodesiense Bioodstream Forms: Surface Ricin-Binding Glycoproteins are Localized Exclusively in the Flagellar Pocket and the Flagellar Adhesion Zone

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.     

Trypanosoma brucei rhodesiense bloodstream forms: surface ricin-binding glycoproteins are localized exclusively in the flagellar pocket and the flagellar adhesion zone

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.     

Cryptobia udonellae sp. n. (Kinetoplastidea: Cryptobiida) - parasites of the excretory system of Udonella murmanica (Udonellida)

A new cryptobiid flagellates, Cryptobia udonellae sp. n., is described from the excretory channels of Udonella murmanica. The body of flagellates is spindle-shaped. The flagellar pocket is subapical. Two flagella emerge from the pocket. One flagellum turns anterior and is forward-directed; the other flagellum is directed posterior and close to the ventral cell surface. The ventral groove is well developed. The cytostome opens just anterior to the flagellar pocket. The cytostome leads to the short cytopharynx. In the excretory channel of worms the flagellates C. udonellae sp. n. are attached to microvilli of epithelium or lay free in the lumen. Both flagellates have been studied with TEM. The unusual parasite system which involves organisms of four different phylums of animals has been described for the first time.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.     

Acylation-dependent and-independent membrane targeting and distinct functions of small myristoylated proteins (SMPs) in Leishmania major

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function.     

Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A

Two carbohydrate-binding probes, the lectin concanavalin A and an anti-carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.     

The Spermatozoon of Brachionus plicatilis(Rotifera,Monogononta) With Some Notes on Sperm Ultrastructure in Rotifera

The spermatozoon of B. plicatilisis a thread–like cell with an anterior flagellar portion and a posterior cell body. The flagellum has a lateral ‘undulating membrane’, containing a folded longitudinal cisterna and an axoneme. The basal body of the axoneme is at the anterior tip. The axoneme lacks outer dynein arms and extends through the entire flagellar region and most of the cell body. The main portion of the flagellum and of the cell body contains a series of vesicles with tightly packed tubules that may serve as a cytoskeleton. The cell body contains a partly condensed nucleus, several mitochondria and some cytoplasm. Some elongated mitochondria are arranged in the postnuclear region. When the spermatozoon moves, the undulations propagate from the basal body at the flagellar tip. Late spermatids can be recognized by the nucleus and the flagellum being coiled and enclosed within a common cell membrane. As in other rotifers, there are cigar–like cell products (‘rods’) in the testes. The general organization of the cell, including the absence of an evident acrosome, resembles that of the other known monogonont sperm types.     

Unusual Structures in the Flagellar Apparatus of a Strain of Trypanosoma cruzi*

Certain structures, associated with the flagellum, and which had hitherto been described as appearing occasionally in some species of trypanosomes, were found very frequently in epimastigote forms of strain F of Trypanosoma cruzi: (a) a group of tubular elements in an electron-dense mass enclosed within a swelling of the flagellar membrane as the flagellum emerges from its reservoir; (b) an expansion of the flagellar membrane at the point of the above swelling, which in cross-sections appears as a ring; and (c) an electron dense band in the body of the organism alongside the border of the flagellar pocket. The possible significance of these structures and the fact that so far they have been found only in one strain of T. cruzi are discussed.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS OF NAEGLERIA

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.     

Host-parasite interactions and trypanosome morphogenesis: a flagellar pocketful of goodies

Trypanosomes are characterised by the possession of a single flagellum and a subpellicular microtubule cytoskeleton. The flagellum is more than an organelle for motility; its position and polarity along with the sub-pellicular cytoskeleton enables the morphogenesis of a distinct flagellar pocket and the flagellar basal body is responsible for positioning and segregating the kinetoplast--the mitochondrial genome. Recent work has highlighted the molecules and morphogenesis of these cytoskeletal/flagellum structures and how dynamic events, occurring in the flagellar pocket and kinetoplast, are critical for host-parasite interactions.     

Surface Domains in the Pathogenic Protozoan Tritrichomonas foetus

ABSTRACT The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus . The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.     

Surface domains in the pathogenic protozoan Tritrichomonas foetus.

The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus. The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.     

Distribution of α-Galactosyl-Containing Epitopes on Trypanosoma cruzi Trypomastigote and Amastigote Forms from Infected Vero Cells Detected by Chagasic Antibodies

Reactivity of different Trypanosoma cruzi developmental forms with purified Chagasic anti-α-galactosyl antibodies (anti-Gal) was studied using epimastigotes from axenic cultures, trypomastigotes and amastigotes from infected Vero cell cultures, and an immunogold labeling method as observed by electron microscopy. Epimastigotes were poorly labeled, whereas extracellular trypomastigotes and amastigotes bound heterogeneously to the antibody with many cells being intensely labeled at the cell surface, including the membrane lining the cell body, the flagellum and the flagellar pocket. Parasites with poor labeling at the cell surface generally had several gold particles within the cell, mostly in cytoplasmic vacuoles. The Golgi complex of trypomastigotes was strongly labeled. Intracellular parasites were labeled at the parasite cell surface or within vacuolar structures. The expression in T. cruzi -infected Vero cells of α-galactosyl antigenic structures acquired from the parasite was shown by moderate labeling with Chagasic anti-Gal of the membrane lining parasite-free outward cell projections. The reactivity with purified anti-Gal from healthy individuals at the same concentrations of Chagasic anti-Gal was poor, with gold particles appearing in the nucleus and cytoplasm but not at the cell surface. It paralleled the labeling with Bandeireae simplicifolia IB-4 lectin. The results provide a basis for autoimmune reactions involving anti-Gal from chronic Chagasic patients.     

Trypanosoma brucei gambiense and T. b. rhodesiense: Concanavalin A Binding to the Membrane and Flagellar Pocket of Bloodstream and Procyclic Forms1

ABSTRACT We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3–16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed >95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.     

The cytology and ultrastructure of zoospores of Hydrurus foetidus (Chrysophyceae)

The unusual tetrahedral shape of Hydrurus foetidus (Vill.) Trev. zoospores is associated with a complex skeletal system of microtubules extending from a broad flagellar root (up to 19 microtubules) into each of three, pointed anterior processes. The posterior end, also pointed and supported by a separate set of microtubules, contains a single large chloroplast with a prominent posterior furrow containing mitochondrial elements. A large immersed pyrenoid is penetrated by paired thylakoids. There is no eyespot. Numerous large Golgi bodies occur immediately anterior to the nucleus and up to 5–6 contractile vacuoles lie near the cell surface at the anterior end. Two terminally inserted flagella extend from the cell surface, a long one serving for cell locomotion, and the other vestigial with an axonemal pattern of 9+0. The flagellar root system consists of: (1) a thin, striated rhizoplast extending from the basal body of the long flagellum and ramifying over the surface of a conspicuous, anteriorly directed, conical projection of the nucleus; (2) a broad microtubular root which emanates from near the basal body of the long flagellum and appears to function as a MTOC; (3) a compound root, consisting of a striated fiber and two associated microtubules, which runs alongside the basal body of the stubby flagellum before terminating at the cell surface; and (4) a short two-membered microtubular root, also associated with the basal body of the stubby flagellum. Other components of the flagellar apparatus include a large dense body near the proximal end of the basal body of the short flagellum, and a small, dense, core-like structure closely associated with one of its triplet fibers. The flagellar apparatus of H. foetidus is remarkably similar in ultrastructure to that of Chrysonebula holmesii Lund.